Small-angle X-ray characterization of the nucleoprotein complexes resulting from DNA-induced oligomerization of HIV-1 integrase

HIV-1 integrase (IN) catalyses integration of a DNA copy of the viral genome into the host genome. Specific interactions between retroviral IN and long terminal repeats (LTR) are required for this insertion. To characterize quantitatively the influence of the determinants of DNA substrate specificity on the oligomerization status of IN, we used the small-angle X-ray scattering (SAXS) technique. Under certain conditions in the absence of ODNs IN existed only as monomers. IN preincubation with specific ODNs led mainly to formation of dimers, the relative amount of which correlated well with the increase in the enzyme activity in the 3′-processing reaction. Under these conditions, tetramers were scarce. Non-specific ODNs stimulated formation of catalytically inactive dimers and tetramers. Complexes of monomeric, dimeric and tetrameric forms of IN with specific and non-specific ODNs had varying radii of gyration (R_g), suggesting that the specific sequence-dependent formation of IN tetramers can probably occur by dimerization of two dimers of different structure. From our data we can conclude that the DNA-induced oligomerization of HIV-1 IN is probably of importance to provide substrate specificity and to increase the enzyme activity.     

Enzymes phosphorylating lipids and polysaccharides

Phosphorylation plays an important role in regulation of living functions of organisms; phosphorylation may significantly alter chemical properties of proteins, lipids, and carbohydrates. Canonical kinases catalyze transfer of terminal phosphate group from ATP (or other NTPs) to specific nucleophilic groups of proteins, lipids, and polysaccharides. Recently, unique kinases, catalytically active antibodies (abzymes) phosphorylating proteins, lipids, and polysaccharides have also been discovered. This review highlights biological functions and enzymatic characteristics of canonical kinases and abzymes phosphorylating lipids and polysaccharides.     

Within‐plant distribution of 1,4‐benzoxazin‐3‐ones contributes to herbivore niche differentiation in maize

Plant defences vary in space and time, which may translate into specific herbivore‐foraging patterns and feeding niche differentiation. To date, little is known about the effect of secondary metabolite patterning on within‐plant herbivore foraging. We investigated how variation in the major maize secondary metabolites, 1,4‐benzoxazin‐3‐one derivatives (BXDs), affects the foraging behaviour of two leaf‐chewing herbivores. BXD levels varied substantially within plants. Older leaves had higher levels of constitutive BXDs while younger leaves were consistently more inducible. These differences were observed independently of plant age, even though the concentrations of most BXDs declined markedly in older plants. Larvae of the well‐adapted maize pest Spodoptera frugiperda preferred and grew better on young inducible leaves irrespective of plant age, while larvae of the generalist Spodoptera littoralis preferred and tended to grow better on old leaves. In BXD‐free mutants, the differences in herbivore weight gain between old and young leaves were absent for both species, and leaf preferences of S. frugiperda were attenuated. In contrast, S. littoralis foraging patterns were not affected. In summary, our study shows that plant secondary metabolites differentially affect performance and foraging of adapted and non‐adapted herbivores and thereby likely contribute to feeding niche differentiation.     

Extremely Stable Soluble High Molecular Mass Multi-Protein Complex with DNase Activity in Human Placental Tissue

Human placenta is an organ which protects, feeds, and regulates the grooving of the embryo. Therefore, identification and characterization of placental components including proteins and their multi-protein complexes is an important step to understanding the placenta function. We have obtained and analyzed for the first time an extremely stable multi-protein complex (SPC, ∼1000 kDa) from the soluble fraction of three human placentas. By gel filtration on Sepharose-4B, the SPC was well separated from other proteins of the placenta extract. Light scattering measurements and gel filtration showed that the SPC is stable in the presence of NaCl, MgCl2, acetonitrile, guanidinium chloride, and Triton in high concentrations, but dissociates efficiently in the presence of 8 M urea, 50 mM EDTA, and 0.5 M NaCl. Such a stable complex is unlikely to be a casual associate of different proteins. According to SDS-PAGE and MALDI mass spectrometry data, this complex contains many major glycosylated proteins with low and moderate molecular masses (MMs) 4–14 kDa and several moderately abundant (79.3, 68.5, 52.8, and 27.2 kDa) as well as minor proteins with higher MMs. The SPC treatment with dithiothreitol led to a disappearance of some protein bands and revealed proteins with lower MMs. The SPCs from three placentas efficiently hydrolyzed plasmid supercoiled DNA with comparable rates and possess at least two DNA-binding sites with different affinities for a 12-mer oligonucleotide. Progress in study of placental protein complexes can promote understanding of their biological functions.     

Antibodies against DNA hydrolyze DNA and RNA

In this work, rabbits were immunized with a high polymer DNA complexed with methylated BSA (mBSA) and by mBSA. It is shown that electrophoretically homogeneous preparations of polyclonal antibodies (Ab) from non-immunized rabbits and animals immunized by mBSA do not exhibit catalytic activity. Ab from the blood of rabbits immunized with the DNA-mBSA complex hydrolyzed poly(C) and different RNAs with efficiency exceeding that towards DNA by approximately 3–4 orders of magnitude. Affinity chromatography of the IgG on DNA cellulose separated the Ab into fractions hydrolyzing both RNA and DNA, and for the first time fractions that hydrolyze only RNA were found. Kinetic parameters that characterize the RNA and DNA hydrolysis by initial Ab preparations and their fractions obtained by separation on an affinity sorbent are compared.     

DNA-hydrolyzing IgG antibodies from the blood of patients with acquired immune deficiency syndrome

DNase activity was analyzed in 110 IgG preparations from the blood of AIDS patients. The relative activity of the preparations varied markedly among patients, being reliably detectable in 96% of the preparations. It was shown with several rigid criteria that DNAase activity is an intrinsic property of antibodies (Abs) from AIDS patients. Not only intact IgG, but also isolated light chains of polyclonal Abs were shown to possess catalytic activity. The abzymes efficiently catalyzed DNA hydrolysis in a wide range of pH (5.0–9.5). The K _M and V _max values were evaluated for Ab-dependent hydrolysis of DNA.     

Immunofluorescent detection of 8-oxoguanine DNA lesions in liver cells from aging OXYS rats, a strain prone to overproduction of free radicals

Production of free radicals in animals is accompanied with a number of pathologic conditions, some of which may be manifested through DNA damage. Studies of mechanisms of oxidative DNA damage by free radicals in vivo are hindered by the lack of good animal models with significant overgeneration of or increased sensitivity to free radicals. An inbred rat strain (OXYS) is characterized by inherited overgeneration of free radicals, lipid peroxidation, protein oxidation, DNA rearrangements, and pathological conditions paralleling several human degenerative diseases. We have used monoclonal antibodies against a common pre-mutagenic base lesion 8-oxoguanine (8-oxoG) in combination with indirect immunofluorescence microscopy and image analysis to follow the relative age-dependent amounts and distribution of 8-oxoG in liver cells from OXYS and Wistar rats. 8-OxoG increased with age in both strains of rats, with OXYS rats always displaying statistically significantly higher levels of oxidative DNA damage than Wistar rats. Statistical analysis indicates that 8-oxoG does not uniformly accumulate in all cells with advancing age or increasing free radical load, but rather concentrates in a minor fraction of cells with a high damage level.     

Ionic strength and magnesium affect the specificity of Escherichia coli and human 8-oxoguanine-DNA glycosylases

An abundant oxidative lesion, 8-oxo-7,8-dihydroguanine (8-oxoG), often directs the misincorporation of dAMP during replication. To prevent mutations, cells possess an enzymatic system for the removal of 8-oxoG. A key element of this system is 8-oxoguanine-DNA glycosylase (Fpg in bacteria, OGG1 in eukaryotes), which must excise 8-oxoG from 8-oxoG:C pairs but not from 8-oxoG:A. We investigated the influence of various factors, including ionic strength, the presence of Mg(2+) and organic anions, polyamides, crowding agents and two small heterocyclic compounds (biotin and caffeine) on the activity and opposite-base specificity of Escherichia coli Fpg and human OGG1. The activity of both enzymes towards 8-oxoG:A decreased sharply with increasing salt and Mg(2+) concentration, whereas the activity on 8-oxoG:C was much more stable, resulting in higher opposite-base specificity when salt and Mg(2+) were at near-physiological concentrations. This tendency was observed with both Cl(-) and glutamate as the major anions in the reaction mixture. Kinetic and binding parameters for the processing of 8-oxoG:C and 8-oxoG:A by Fpg and OGG1 were determined under several different conditions. Polyamines, crowding agents, biotin and caffeine affected the activity and specificity of Fpg or OGG1 only marginally. We conclude that, in the intracellular environment, the specificity of Fpg and OGG1 for 8-oxoG:C versus 8-oxoG:A is mostly due to high ionic strength and Mg(2+).     

Effects of umbilical cord blood stem cells on healing factors for diabetic foot injuries

The use of stem or progenitor cells from bone marrow, or peripheral or umbilical cord blood is becoming more common for treatment of diabetic foot problems. These cells promote neovascularization by angiogenic factors and they promote epithelium formation by stimulating cell replication and migration under certain pathological conditions. We investigated the role of CD34 + stem cells from human umbilical cord blood in wound healing using a rat model. Rats were randomly divided into a control group and two groups with diabetes induced by a single dose of 55 mg/kg intraperitoneal streptozocin. Scarred areas 5 mm in diameter were created on the feet of all rats. The diabetic rats constituted the diabetes control group and a diabetes + stem cell group with local injection into the wound site of 0.5 × 106 CD34 + stem cells from human umbilical cord blood. The newly formed skin in the foot wounds following CD34 + stem cell treatment showed significantly improvement by immunohistochemistry and TUNEL staining, and were closer to the wound healing of the control group than the untreated diabetic animals. The increase in FGF expression that accompanied the local injection of CD34 + stem cells indicates that FGF stimulation helped prevent apoptosis. Our findings suggest a promising new treatment approach to diabetic wound healing.