Structure of (dG)n.(dC)n under superhelical stress and acid pH

We have recently shown that a (GA)n.(TC)n tract undergoes a sharp structural transition under superhelical stress (V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 2,327 (1985]. Unlike the well studied transitions to the cruciform and to the Z form, this novel transition was strongly pH-dependent. We have found the (dG)n.(dC)n insert to undergo a pH-dependent structural transition similar to that of the (GA)n.(TC)n tract. These new data meet our earlier expectations and disagree with the data of D.E. Pulleyblank, D.B. Haniford and A.R. Morgan, Cell 42, 271 (1985). We conclude that a novel DNA structure (the H-form) is typical of homopurine-homopyrimidine mirror repeats (the H palindromes) under superhelical stress and/or acid pH. In the H-form the homopyrimidine strand forms a hairpin while half of the homopurine strand interacts with the hairpin forming a triplex, the other half of the homopurine strand being unstructured (V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 2, 3, 667 (1986].     

Formation of intramolecular triplex in homopurine-homopyrimidine mirror repeats with point substitutions.

We have used two-dimensional gel electrophoresis to study the structural transition to the triplex H form of sequences 5'-AAGGGAGAAXGGGGTATAGGGGYAAGAGGGAA-3' where X and Y are any DNA bases. The transition was observed at acid pH under superhelical stress. For X = Y = A or X = Y = G the sequences corresponded to homopurine-homopyrimidine mirror repeats (H-palindrome) which are known to adopt the H form under acid pH and superhelical stress. We have shown that the H form is actually formed for all X and Y, though in cases other than X = Y = A and X = Y = G the transition requires larger negative superhelical stress. Different substitutions require different superhelicity levels for the transition to occur. Theoretical analysis of the data obtained made it possible to estimate the energy cost of triplex formation due to all possible mismatched base triads.     

A structural transition in d(AT)n.d(AT)n inserts within superhelical DNA

We have constructed plasmids carrying d(AT)n.d(AT)n inserts of different lengths. Two-dimensional gel electrophoresis patterns show that an increase in the negative superhelicity of these DNAs brings about a structural transition within the inserts, resulting in a reduction of the superhelical stress. However, this reduction corresponds to the expected values neither for cruciform nor the Z form. Those DNA topoisomers in which the structural transition had occurred proved to be specifically recognizable by single-strand-specific endonuclease S1, with the cleavage site situated at the centre of the insert. These data, as well as kinetic studies, suggest that the cloned d(AT)n.d(AT)n sequences adopt a cruciform rather than the Z-form structure. We discuss plausible reasons of the discrepancy between the observed superhelical stress release and that expected for the transition of the insert to the cruciform state.     

Structures of homopurine-homopyrimidine tract in superhelical DNA

For homopurine-homopyrimidine tracts in superhelical DNA, we propose a structure involving Watson-Crick and Hoogsteen paired triple helixes, hairpin loops and unstructured domains. Topologically, the whole structure is equivalent to an open region. The proposed structure is consistent with available S1 cleavage, pH and alkylation data and energetics under superhelical stress; this new structure is a much more probable candidate than the one proposed by us recently (V.I. Lyamichev, S.M. Mirkin & M.D. Frank-Kamenetskii, J. Biomole. Str. Dyns 3, 327-338, 1985).     

Topography of the gap junctions in human skin and their possible role in the nonneural transmission of information

Localization and distribution of the gap junctions in the human epidermis have been studied. They are mainly concentrated in the area of biologically active (acupunctive) points and in so called meridians connecting these points. A supposition is made that the gap junction system performs certain integration of the information from the skin surface and its aneural transmission to remote areas. Discovery of a regulated gap junction system in relatively low organized animals (previously described by the authors), as well as revealing of such a system in mammals, makes it possible to suppose that the system is phylogenetically the most ancient one performing a directed transmission of an information simultaneously with and besides the neural system.     

Native supercoiling of DNA: The effects of DNA gyrase and ω protein in E. coli

Molecular Genetics and Genomics - This study deals with the effects of a temperature-sensitive (ts) mutation at the gene encoding the DNA gyrase B subunit (gyrB ts) and a deletion of the top gene...     

Biological maturation and β-adrenergic effectors: development of β-adrenergic receptors in rabbit heart

Summary The -adrenergic receptor, transduction processes and catalytic activity of the adenylate cyclase enzyme complex have been investigated in rabbit heart at different stages of biological maturation. The binding of [³H]-dihydroalprenolol to a washed membrane preparation isolated from rabbit ventricular muscle was used to characterize -adrenergic receptors. Significant age-related differences were noted in -receptor affinity (K_d) and density (RD) of neonatal and adult animals; the adult K_d was 3.7-fold greater and the RD 2-fold higher than the neonates. No significant differences in these parameters were detected among the 27-day old fetus and the 1- and 7-day old neonates. Age-dependent differences in agonist isoproterenol affinity for the receptor were not observed in contrast to the significant changes in antagonist (DHA) affinity.Age-related changes in receptor affinity were also quantitated by determining the inhibitory potency of alprenolol on isoproterenol stimulated adenylate cyclase enzyme activity. A decreased affinity of the -adrenergic receptor for alprenolol in the adult heart was indicated by a 3.7-fold greater K_i for the adult than the 1-day old neonate. Ontogenic variations in the coupling efficiency between the receptor and catalytic components of the adenylate cyclase complex were also evaluated. The K_d of the -adrenergic receptor for isoproterenol and the EC50 for adenylate cyclase stimulation were determined under similar conditions. The corresponding coupling index (K_d/EC50) was found to be 2.4-fold greater in the 1-day old neonate than adult, suggesting that for a given percentage increase in adenylate cyclase activity, a lower percentage of -adrenergic receptor sites need be occupied in the neonate. These data extend previous studies (29) and indicate all components of the rabbit heart adenylate cyclase enzyme complex (i.e., the -adrenergic receptor, the GTP-dependent transduction event, and the catalytic subunit) exhibit significant developmental changes.     

IL-10 Inhibits the NF-κB and ERK/MAPK-Mediated Production of Pro-Inflammatory Mediators by Up-Regulation of SOCS-3 in Trypanosoma cruzi-Infected Cardiomyocytes

Trypanosoma cruzi (T. cruzi) infection produces an intense inflammatory response which is critical for the control of the evolution of Chagas’ disease. Interleukin (IL)-10 is one of the most important anti-inflammatory cytokines identified as modulator of the inflammatory reaction. This work shows that exogenous addition of IL-10 inhibited ERK1/2 and NF-κB activation and reduced inducible nitric oxide synthase (NOS2), metalloprotease (MMP) -9 and MMP-2 expression and activities, as well as tumour necrosis factor (TNF)-α and interleukin (IL)-6 expression, in T. cruzi-infected cardiomyocytes. We found that T. cruzi and IL-10 promote STAT3 phosphorylation and up-regulate the expression of suppressor of cytokine signalling (SOCS)-3 thereby preventing NF-κB nuclear translocation and ERK1/2 phosphorylation. Specific knockdown of SOCS-3 by small interfering RNA (siRNA) impeded the IL-10-mediated inhibition of NF-κB and ERK1/2 activation. As a result, the levels of studied pro-inflammatory mediators were restored in infected cardiomyocytes. Our study reports the first evidence that T. cruzi up- regulates SOCS-3 expression and highlights the relevance of IL-10 in the modulation of pro-inflammatory response of cardiomyocytes in Chagas’ disease.     

Lung tissue extracellular matrix-derived hydrogels protect against radiation-induced lung injury by suppressing epithelial–mesenchymal transition

This study aimed to examine whether lung tissue extracellular matrix (ECM) hydrogels have protective effects on radiation-induced lung injury (RILI). The cytocompatibility and histocompatibility were tested for the obtained ECM-derived hydrogel. Sprague–Dawley rats were randomly divided into three groups (n = 18): control group (control); rats receiving irradiation and intratracheal injection of normal saline (IR + NS); and rats receiving irradiation and intratracheal injection of lung ECM-derived hydrogel (IR + ECM). The wet/dry weight ratio was used to evaluate the congestion and edema of the lungs. Histopathological analysis of lung tissues was performed using hemotoxylin and eosin staining and Masson's trichrome staining. Immunohistochemical staining and western blot analyses were carried out to determine the expression of epithelial–mesenchymal transition (EMT)-related proteins in lung tissues (E-cadherin, α-smooth muscle actin [α-SMA], and vimentin). In addition, tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6), hydroxyproline, malondialdehyde (MDA), and superoxide dismutase (SOD) levels were also evaluated. The ECM-derived hydrogels had good cytocompatibility and histocompatibility. ECM-derived hydrogel treatment improved lung histopathology injury and pulmonary edema. Higher expression of E-cadherin and lower expression of vimentin and α-SMA were found in the IR + ECM group compared with those in the IR + NS group. Hydroxyproline levels were reduced by ECM-derived hydrogel treatment compared with those in the IR + NS group. Obvious increases of TNF-α, IL-6, and TGF-β1 were identified following irradiation. Marked reductions in MDA content and increases in SOD were induced by ECM-derived hydrogel treatment in rats after radiation. ECM-derived hydrogels were shown to protect against RILI, potentially by reducing EMT, inflammation, and oxidative damage.