The Mechanism of Supercoiled DNA Recognition by Eukaryotic Type I Topoisomerases: II. A Comparison of the Enzyme Interaction with Specific and Nonspecific Oligonucleotides

Data on the interaction of DNA type I topoisomerases from the murine and human placenta cells with specific and nonspecific oligonucleotides of various structures and lengths are summarized. The relative contributions of various contacts between the enzymes and DNA that have previously been detected by X-ray analysis to the total affinity of the topoisomerases for DNA substrates are estimated. Factors that determine the differences in the enzyme interactions with specific and nonspecific single- and double-stranded DNAs are revealed. The results of the X-ray analysis of human DNA topoisomerase I are interpreted taking into account data on the comprehensive thermodynamic and kinetic analysis of the enzyme interaction with the specific and nonspecific DNAs.     

Human milk lactoferrin binds two DNA molecules with different affinities.

Evidence is presented that lactoferrin (LF), an Fe3+-binding glycoprotein, possesses two DNA-binding sites with different affinities for specific oligonucleotides (ODNs) (Kdl = 8 nM; Kd2 approximately 0.1 mM). The high affinity site became labeled after incubation with affinity probes for DNA-binding sites; like the antibacterial and polyanion-binding sites, this site was shown to be located in the N-terminal domain of LF. Interaction of heparin with the polyanion-binding site inhibits the binding of ODNs to both sites. These data suggest that the DNA-binding sites of LF coincide or overlap with the known polyanion and antimicrobial domains of the protein.     

Specific anti‐integrase abzymes from HIV‐infected patients: a comparison of the cleavage sites of intact globular HIV integrase and two 20‐mer oligopeptides corresponding to its antigenic determinants

HIV‐infected patients possess anti‐integrase (IN) IgGs and IgMs that, after isolation by chromatography on IN‐Sepharose, unlike canonical proteases, specifically hydrolyze only IN but not many other tested proteins. Hydrolysis of intact globular IN first leads to formation of many long fragments of protein, while its long incubation with anti‐IN antibodies, especially in the case of abzymes (Abzs) with a high proteolytic activity, results in the formation of short and very short oligopeptides (OPs). To identify all sites of IgG‐mediated proteolysis corresponding to known AGDs of integrase, we have used a combination of reverse‐phase chromatography, matrix‐assisted laser desorption/ionization spectrometry, and thin‐layer chromatography to analyze the cleavage products of two 20‐mer OPs corresponding to these AGDs. Both OPs contained 9–10 mainly clustered major, medium, and minor sites of cleavage. The main superficial cleavage sites of the AGDs in the intact IN and sites of partial or deep hydrolysis of the peptides analyzed do not coincide. The active sites of anti‐IN Abzs are localized on their light chains, whereas the heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of Abzs provide high specificity of IN hydrolysis. The affinity of anti‐IN Abzs for intact integrase was ~1000‐fold higher than for the OPs. The data suggest that both OPs interact mainly with the light chains of different monoclonal Abzs of the total pool of IgGs, which possesses lower affinity for substrates; and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific and remarkably different in comparison with the cleavage of intact globular IN. Copyright © 2013 John Wiley & Sons, Ltd.     

Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein

Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP‐Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26–27 kDa). Seventy‐two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty‐two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7–9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease‐like and three thiol protease‐like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca²⁺, Mg²⁺_, Mn²⁺, Ni²⁺, Zn²⁺, Cu²⁺, and Co²⁺ was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti‐MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti‐MBP abzymes, which can attack MBP of myelin‐proteolipid sheath of axons and play an important role in MS and SLE pathogenesis. Copyright © 2015 John Wiley & Sons, Ltd.     

Diversity of DNA‐hydrolyzing antibodies from the sera of autoimmune‐prone MRL/MpJ‐lpr mice

It has been shown for the first time that polyclonal IgG abzymes (Abzs) with DNase activity from the sera of autoimmune‐prone MRL/MpJ‐lpr mice can be separated by isoelectric focusing into many subfractions having the isoelectric points (pI) from 4.5 to 9, with the maximal activity for Abzs with pI = 6.5–9.0. Affinity chromatography on DNA‐cellulose separated DNase IgGs into many subfractions demonstrating a range of affinities for DNA and different levels of the relative DNase activities (RDA) due to intrinsically bound metals and after addition of external Mg²⁺, Mn²⁺, Ca²⁺, and Mg²⁺+Ca²⁺. Some fractions significantly increase RDAs in the presence of external ions (Mg²⁺+Ca²⁺ > Mg²⁺ > Mn²⁺ > Ca²⁺), while each of this cofactor can also inhibit or have no influence on the RDAs of another fractions. It is known that complexes of DNA with histones and other proteins of apoptotic cells are the primary immunogens in systemic lupus erythematosus (SLE). Bovine serum albumin (BSA) and methylated BSA (mBSA) increase the RDAs of only some fractions, while have no effect or inhibit other IgG fractions. The ratio of the RDAs in the presence of all metal ions, BSA, and mBSA was individual for every abzyme fraction. Mn²⁺ and Ca²⁺ stimulated accumulation of only relaxed form of supercoiled DNA (scDNA) in the case of all subfractions, while in the presence of Mg²⁺ antibodies (Abs) of some subfractions (and in the presence of Mn²⁺ +Ca²⁺ all subfractions) produced relaxed DNA (rDNA) and linear DNA (linDNA) in a variable extent. The data obtained show that the polyclonal Abzs of mice may be a cocktail of Abs directly to DNA, RNA, and their complexes with proteins and anti‐idiotypic Abs to active centers of different nucleases. The diversity of the physicochemical and kinetic characteristics of the Abzs seems to be significantly widened when pre‐diseased mice spontaneously develop the disease. Copyright © 2010 John Wiley & Sons, Ltd.     

马鹿茸血酶解肽体内免疫功能及抗氧化功能关系的研究

本文分别选用由木瓜蛋白酶水解天山马鹿茸血获得的肽Ⅰ、肽Ⅱ以及原血为受试样品,研究天山马鹿茸血及其酶解肽对小鼠免疫功能和抗氧化功能的影响,其中肽Ⅰ、肽ⅡDH分别为18．1％、12．2％时,清除OH·能力最强,清除率为50．8％。分别测定了脏器指数,脾细胞增殖实验,血清中溶血素和抗体生成细胞的含量以及小鼠血清总抗氧化能力、SOD活力和MDA含量。结果表明：与对照组相比较,肽Ⅱ组能极显著提高小鼠体液和细胞免疫功能,加强小鼠的抗氧化功能（P〈0．01或P〈0．05）。此外,具有较强抗氧化活性的肽显示出了很强的免疫活性（P〈0．05）。     

Catalytic antibodies in the bone marrow and other organs of Th mice during spontaneous development of experimental autoimmune encephalomyelitis associated with cell differentiation

Exact mechanisms of autoimmune disease development are still yet unknown. However, it is known that the development of autoimmune diseases is associated with defects in the immune system, namely, the violation of the bone marrow hematopoietic stem cells (HSCs) differentiation profiles. Different characteristics of autoimmune reaction development in experimental autoimmune encephalomyelitis (EAE) prone Th mice characterizing T-lymphocytes response were analyzed using standard approaches. Profiles of several HSCs differentiation of bone marrow (BFU-E, CFU-E, CFU-GM, CFU-GEMM, T- and B-lymphocytes) of Th male and female mice during spontaneous development of EAE were noticeably different. Patterns of total lymphocytes, B- and T-cells proliferation in several different organs (bone marrow, blood, spleen, thymus, and lymph nodes) were also remarkably different. In addition, there were in time noticeable differences in their changes for some organs of male and female mice. Characters of changes in the profiles of CD4 and CD8 cells proliferation in some organs not always coincide with those for total T lymphocytes. The changes in the differentiation profiles of HSCs and the level of lymphocytes proliferation in the bone marrow and other organs were associated with the increase in the concentration of antibodies against DNA, myelin basic protein, and myelin oligodendrocyte glycoprotein, and catalytic antibodies hydrolyzing these substrates. Despite some differences in changes in the analyzed parameters, in general, the spontaneous development of EAE in male and female mice occurs to some extent in a comparable way.

     

Blood Biochemistry of Sea Turtles Captured in Gillnets in the Lower Cape Fear River,North Carolina,USA

ABSTRACT Mortality due to fisheries interactions has been implicated as a contributor to population decline for several species of sea turtle. The incidental capture of sea turtles in the coastal gillnet fisheries of North Carolina, USA, has received much attention in recent years, and mitigation measures to reduce sea turtle mortality due to gillnet entanglement are a high priority for managers and conservationists. Efforts to evaluate effects of gillnet entanglement on sea turtle populations are complicated by the lack of information on health status of turtles released alive from nets and postrelease mortality. We obtained blood samples from green (Chelonia mydas) and Kemp's ridley (Lepidochelys kempii) sea turtles captured in gillnets for 20–240 minutes to assess the impacts of gillnet entanglement on blood biochemistry and physiological status. We measured concentrations of lactate, corticosterone, ions (Na⁺, K⁺, Cl^-, P, Ca²⁺), enzymes (lactate dehydrogenase [LDH], creatine phosphokinase [CPK], aspartate aminotransferase [AST]), protein, and glucose in the blood and also performed physical examinations of turtles to document external indicators of health status (injuries, lethargy, muted reflexes). We evaluated the effects of entanglement time on blood biochemistry and to look for correlations between blood biochemistry and results of the physical examinations. We observed a significant increase in blood lactate, LDH, CPK, phosphorus, and glucose with increased entanglement time. Alterations in blood biochemistry were generally associated with a decline in health status as indicated by results of the physical examination. Although entanglement time plays an important role in determining the health status of sea turtles upon release from a gillnet, our results suggest that factors such as the depth and severity of entanglement may also have an effect on health status of turtles and the probability of postrelease survival. We were unable to set a maximum unattended gillnet soak time to minimize impacts on captured sea turtles, and therefore recommend that fisheries managers continue to enforce the net attendance regulations currently in place in the lower Cape Fear River, North Carolina, during the summer months.     

Heavy metal ions affect the activity of DNA glycosylases of the Fpg family

Prokaryotic enzymes formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei) and their eukaryotic homologs NEIL1, NEIL2, and NEIL3 define the Fpg family of DNA glycosylases, which initiate the process of repair of oxidized DNA bases. The repair of oxidative DNA lesions is known to be impaired in vivo in the presence of ions of some heavy metals. We have studied the effect of salts of several alkaline earth and transition metals on the activity of Fpg-family DNA glycosylases in the reaction of excision of 5,6-dihydrouracil, a typical DNA oxidation product. The reaction catalyzed by NEIL1 was characterized by values K _m = 150 nM and k _cat = 1.2 min⁻¹, which were in the range of these constants for excision of other damaged bases by this enzyme. NEIL1 was inhibited by Al³⁺, Ni²⁺, Co²⁺, Cd²⁺, Cu²⁺, Zn²⁺, and Fe²⁺ in Tris-HCl buffer and by Cd²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ in potassium phosphate buffer. Fpg and Nei, the prokaryotic homologs of NEIL1, were inhibited by the same metal ions as NEIL1. The values of I₅₀ for NEIL1 inhibition were 7 μM for Cd²⁺, 16 μM for Zn²⁺, and 400 μM for Cu²⁺. The inhibition of NEIL1 by Cd²⁺, Zn²⁺, and Cu²⁺ was at least partly due to the formation of metal-DNA complexes. In the case of Cd²⁺ and Cu²⁺, which preferentially bind to DNA bases rather than phosphates, the presence of metal ions caused the enzyme to lose the ability for preferential binding to damaged DNA. Therefore, the inhibition of NEIL1 activity in removal of oxidative lesions by heavy metal ions may be a reason for their comutagenicity under oxidative stress.