Stability and DNA binding ability of the DNA binding domains of interferon regulatory factors 1 and 3

The thermodynamic properties and DNA binding ability of the N-terminal DNA binding domains of interferon regulatory factors IRF-1 (DBD1) and IRF-3 (DBD3) were studied using microcalorimetric and optical methods. DBD3 is significantly more stable than DBD1: at 20 degrees C the Gibbs energy of unfolding of DBD3 is -28.6 kJ/mol, which is 2 times larger than that of DBD1, -14.9 kJ/mol. Fluorescence anisotropy titration experiments showed that at this temperature the association constants with the PRDI binding site are 1.1 x 10(6) M(-)(1) for DBD1 and 3.6 x 10(6) M(-)(1) for DBD3, corresponding to Gibbs energies of association of -34 and -37 kJ/mol, respectively. However, the larger binding energy of DBD3 is due to its larger electrostatic component, while its nonelectrostatic component is smaller than that of DBD1. Therefore, DBD1 appears to have more sequence specificity than DBD3. Binding of DBD1 to target DNA is characterized by a substantially larger negative enthalpy than binding of DBD3, implying that the more flexible structure of DBD1 forms tighter contacts with DNA than the more rigid structure of DBD3. Thus, the strength of the DBDs' specific association with DNA is inversely related to the stability of the free DBDs.     

Effects of the HIV Protease Inhibitor Ritonavir on GLUT4 Knock-out Mice

HIV protease inhibitors acutely block glucose transporters (GLUTs) in vitro, and this may contribute to altered glucose homeostasis in vivo. However, several GLUT-independent mechanisms have been postulated. To determine the contribution of GLUT blockade to protease inhibitor-mediated glucose dysregulation, the effects of ritonavir were investigated in mice lacking the insulin-sensitive glucose transporter GLUT4 (G4KO). G4KO and control C57BL/6J mice were administered ritonavir or vehicle at the start of an intraperitoneal glucose tolerance test and during hyperinsulinemic-euglycemic clamps. G4KO mice exhibited elevated fasting blood glucose compared with C57BL/6J mice. Ritonavir impaired glucose tolerance in control mice but did not exacerbate glucose intolerance in G4KO mice. Similarly, ritonavir reduced peripheral insulin sensitivity in control mice but not in G4KO mice. Serum insulin levels were reduced in vivo in ritonavir-treated mice. Ritonavir reduced serum leptin levels in C57BL/6J mice but had no effect on serum adiponectin. No change in these adipokines was observed following ritonavir treatment of G4KO mice. These data confirm that a primary effect of ritonavir on peripheral glucose disposal is mediated through direct inhibition of GLUT4 activity in vivo. The ability of GLUT4 blockade to contribute to derangements in the other molecular pathways that influence insulin sensitivity remains to be determined.     

Electrostatic properties of promoter recognized by E. coli RNA polymerase Esigma70

A comparative analysis of electrostatic patterns for 359 sigma70-specific promoters and 359 nonpromoter regions on electrostatic map of Escherichia coli genome was carried out. It was found that DNA is not a uniformly charged molecule. There are some local inhomogeneities in its electrostatic profile which correlate with promoter sequences. Electrostatic patterns of promoter DNAs can be specified due to the presence of some distinctive motifs which differ for different promoter groups and may be involved as signal elements in differential recognition of various promoters by the enzyme. Some specific electrostatic elements which are responsible for modulating promoter activities due to ADP-ribosylation of RNA polymerase alpha-subunit were found in far upstream regions of T4 phage early promoters and E. coli ribosomal promoters.     

New stochastic carcinogenesis model with covariates: an approach involving intracellular barrier mechanisms

In this paper we present a new multiple-pathway stochastic model of carcinogenesis with potential of predicting individual incidence risks on the basis of biomedical measurements. The model incorporates the concept of intracellular barrier mechanisms in which cell malignization occurs due to an inefficient operation of barrier cell mechanisms, such as antioxidant defense, repair systems, and apoptosis. Mathematical formalism combines methodological innovations of mechanistic carcinogenesis models and stochastic process models widely used in studying biodemography of aging and longevity. An advantage of the modeling approach is in the natural combining of two types of measures expressed in terms of model parameters: age-specific hazard rate and means of barrier states. Results of simulation studies allow us to conclude that the model parameters can be estimated in joint analyses of epidemiological data and newly collected data on individual biomolecular measurements of barrier states. Respective experimental designs for such measurements are suggested and discussed. An analytical solution is obtained for the simplest design when only age-specific incidence rates are observed. Detailed comparison with TSCE model reveals advantages of the approach such as the possibility to describe decline in risk at advanced ages, possibilities to describe heterogeneous system of intermediate cells, and perspectives for individual prognoses of cancer risks. Application of the results to fit the SEER data on cancer risks demonstrates a strong predictive power of the model. Further generalizations of the model, opportunities to measure barrier systems, biomedical and mathematical aspects of the new model are discussed.     

Scar tissue classification using nonlinear optical microscopy and discriminant analysis

This paper addresses the scar tissue maturation process that occurs stepwise, and calls for reliable classification. The structure of collagen imaged by nonlinear optical microscopy (NLOM) in post‐burn hypertrophic and mature scar, as well as in normal skin, appeared to distinguish these maturation steps. However, it was a discrimination analysis, demonstrated here, that automated and quantified the scar tissue maturation process. The achieved scar classification accuracy was as high as 96%. The combination of NLOM and discrimination analysis is believed to be instrumental in gaining insight into the scar formation, for express diagnosis of scar and surgery planning. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Low-frequency component of photoplethysmogram reflects the autonomic control of blood pressure

The question of how much information the photoplethysmogram (PPG) signal contains on the autonomic regulation of blood pressure (BP) remains unsolved. This study aims to compare the low-frequency (LF) and high-frequency components of PPG and BP and assess their correlation with oscillations in interbeat (RR) intervals at similar frequencies. The PPG signal from the distal phalanx of the right index finger recorded using a reflective PPG sensor at green light, the BP signal from the left hand recorded using a Finometer, and RR intervals were analyzed. These signals were simultaneously recorded within 15 min in a supine resting condition in 17 healthy subjects (12 males and 5 females) aged 33 ± 9 years (mean ± SD). The study revealed the high coherence of LF components of PPG and BP with the LF component of RR intervals. The high-frequency components of these signals had low coherence. The analysis of the signal instantaneous phases revealed the presence of high-phase coherence between the LF components of PPG and BP. It is shown that the LF component of PPG is determined not only by local myogenic activity but also reflects the processes of autonomic control of BP.     

Solution structure of the R3H domain from human Smubp-2

The R3H domain is a conserved sequence motif, identified in over 100 proteins, that is thought to be involved in polynucleotide-binding, including DNA, RNA and single-stranded DNA. In this work the 3D structure of the R3H domain from human Smubp-2 was determined by NMR spectroscopy. It is the first 3D structure determination of an R3H domain. The fold presents a small motif, consisting of a three-stranded antiparallel beta-sheet and two alpha-helices, which is related to the structures of the YhhP protein and the C-terminal domain of the translational initiation factor IF3. The similarities are non-trivial, as the amino acid identities are below 10%. Three conserved basic residues cluster on the same face of the R3H domain and could play a role in nucleic acid recognition. An extended hydrophobic area at a different site of the molecular surface could act as a protein-binding site. A strong correlation between conservation of hydrophobic amino acids and side-chain solvent protection indicates that the structure of the Smubp-2 R3H domain is representative of R3H domains in general.     

Mismatch repair proteins are activators of toxic responses to chromium-DNA damage

Chromium(VI) is a toxic and carcinogenic metal that causes the formation of DNA phosphate-based adducts. Cr-DNA adducts are genotoxic in human cells, although they do not block replication in vitro. Here, we report that induction of cytotoxicity in Cr(VI)-treated human colon cells and mouse embryonic fibroblasts requires the presence of all major mismatch repair (MMR) proteins. Cr-DNA adducts lost their ability to block replication of Cr-modified plasmids in human colon cells lacking MLH1 protein. The presence of functional mismatch repair caused induction of p53-independent apoptosis associated with activation of caspases 2 and 7. Processing of Cr-DNA damage by mismatch repair resulted in the extensive formation of gamma-H2AX foci in G(2) phase, indicating generation of double-stranded breaks as secondary toxic lesions. Induction of gamma-H2AX foci was observed at 6 to 12 h postexposure, which was followed by activation of apoptosis in the absence of significant G(2) arrest. Our results demonstrate that mismatch repair system triggers toxic responses to Cr-DNA backbone modifications through stress mechanisms that are significantly different from those for other forms of DNA damage. Selection for Cr(VI) resistant, MMR-deficient cells may explain the very high frequency of lung cancers with microsatellite instability among chromate workers.     

Polynuclear water-soluble dinitrosyl iron complexes with cysteine or glutathione ligands: Electron paramagnetic resonance and optical studies

Electron paramagnetic resonance and optical spectrophotometric studies have demonstrated that low-molecular dinitrosyl iron complexes (DNICs) with cysteine or glutathione exist in aqueous solutions in the form of paramagnetic mononuclear (М-DNICs) and diamagnetic binuclear complexes (B-DNICs). The latter represent Roussin’s red salt esters and can be prepared by treatment of aqueous solutions of Fe²⁺ and thiols (рН 7.4) with gaseous nitric oxide (NO) at the thiol:Fe²⁺ ratio 1:1. М-DNICs are synthesized under identical conditions at the thiol:Fe²⁺ ratios above 20 and produce an EPR signal with an electronic configuration {Fe(NO)₂}⁷ at g_aver. = 2.03. At neutral pH, aqueous solutions contain both M-DNICs and B-DNICs (the content of the latter makes up to 50% of the total DNIC pool). The concentration of B-DNICs decreases with a rise in pH; at рН 9–10, the solutions contain predominantly M-DNICs. The addition of thiol excess to aqueous solutions of B-DNICs synthesized at the thiol:Fe²⁺ ratio 1:2 results in their conversion into М-DNICs, the total amount of iron incorporated into M-DNICs not exceeding 50% of the total iron pool in B-DNICs. Air bubbling of cys-М-DNIC solutions results in cysteine oxidation-controlled conversion of М-DNICs first into cys-B-DNICs and then into the EPR-silent compound Х able to generate a strong absorption band at 278 nm. In the presence of glutathione or cysteine excess, compound Х is converted into B-DNIC/M-DNIC and is completely decomposed under effect of the Fe²⁺ chelator о-phenanthroline or N-methyl-d-glucamine dithiocarbamate (MGD). Moreover, MGD initiates the synthesis of paramagnetic mononitrosyl iron complexes with MGD. It is hypothesized that compound Х represents a polynuclear DNIC with cysteine, most probably, an appropriate Roussin’s black salt thioesters and cannot be prepared by simple substitution of М-DNIC cysteine for glutathione. Treatment of М-DNIC with sodium dithionite attenuates the EPR signal at g_aver. = 2.03 and stimulates the appearance of an EPR signal at g_aver. = 2.0 with a hypothetical electronic configuration {Fe(NO)₂}⁹. These changes can be reversed by storage of DNIC solutions in atmospheric air. The EPR signal at g_aver. = 2.0 generated upon treatment of B-DNICs with dithionite also disappears after incubation of B-DNIC solutions in air. In all probability, the center responsible for this EPR signal represents М-DNIC formed in a small amount during dithionite-induced decomposition of B-DNIC.