Characteristics of monoclonal antibodies against human thyroid peroxidase for use in immunoaffinity chromatography and immunoassays

Two types of monoclonal antibodies (MABs) against human thyroid peroxidase (TPO) have been obtained, which interact with spatially separated conformational epitopes of the antigen (Ka values are in the range 10(8)-10(9) M(-1)). The binding site of MAB F8 is in the immunodominant region of the TPO molecule, in the vicinity of the autoantigenic determinants, whereas the epitope specific for MAB A1 lies outside this location. Both MABs retain the ability to form immune complexes after solid-phase immobilization and chemical modification with a biotin derivative. The above properties suggest that MABs A1 and F8 may be used in immunoaffinity chromatography (isolation and purification of TPO from natural sources) and immunoassays for determinations of TPO (in biological fluids) and TPO autoantibodies (in human blood serum).     

Immunoaffinity chromatography of human thyroid peroxidase: The stability of the three-dimensional structure and immunoreactivity of antigen and antibodies under various elution conditions

Actions of various chemical agents modeling immunoaffinity chromatography elution conditions caused structural changes of the components of human thyroid peroxidase (TPO) complexes with monoclonal antibodies (MABs) F8 and A1 whose antigenic determinants have a conformational nature and are located in the immunodominant region and a peripheral region of TPO, respectively. These changes became apparent in the circular dichroism and fluorescence spectra of TPO and both MABs as well as in the immunoassay. The effectiveness of the chemical reagents with respect to TPO desorption from an immobilized MAB decreased in the following order: 0.2 M ammonia (pH 11.5) > 0.1 M lithium 3,5-diiodosalycilate > 0.1 M glycine-HCl (pH 2.5) > 1 M NaI > 30% propylene glycol + 1 M NaCl > 30% propylene glycol > 1 M NaCl. At pH 11.5, the three-dimensional structure and immunoreactivity of TPO retained completely and only minor alterations of MAB analogical parameters took place, thus providing a high yield of the functional active human TPO and favoring repeated use of the immobilized MABs in immunoaffinity chromatography. The results may be used as a strategy for the optimization of various protein antigens immunoaffinity chromatography.     

Imaging Striatal Microglial Activation in Patients with Parkinson’s Disease

This study investigated whether the second-generation translocator protein 18kDa (TSPO) radioligand, [¹⁸F]-FEPPA, could be used in neurodegenerative parkinsonian disorders as a biomarker for detecting neuroinflammation in the striatum. Neuroinflammation has been implicated as a potential mechanism for the progression of Parkinson’s disease (PD). Positron Emission Tomography (PET) radioligand targeting for TSPO allows for the quantification of neuroinflammation in vivo. Based on genotype of the rs6791 polymorphism in the TSPO gene, 16 mixed-affinity binders (MABs) (8 PD and age-matched 8 healthy controls (HCs)), 16 high-affinity binders (HABs) (8 PD and age-matched 8 HCs) and 4 low-affinity binders (LABs) (3 PD and 1 HCs) were identified. Total distribution volume (V_T) values in the striatum were derived from a two-tissue compartment model with arterial plasma as an input function. There was a significant main effect of genotype on [¹⁸F]-FEPPA V_T values in the caudate nucleus (p = 0.001) and putamen (p < 0.001), but no main effect of disease or disease x genotype interaction in either ROI. In the HAB group, the percentage difference between PD and HC was 16% in both caudate nucleus and putamen; in the MAB group, it was -8% and 3%, respectively. While this PET study showed no evidence of increased striatal TSPO expression in PD patients, the current findings provide some insights on the possible interactions between rs6791 polymorphism and neuroinflammation in PD.     

Notch signaling regulates myogenic regenerative capacity of murine and human mesoangioblasts

Somatic stem cells hold attractive potential for the treatment of muscular dystrophies (MDs). Mesoangioblasts (MABs) constitute a myogenic subset of muscle pericytes and have been shown to efficiently regenerate dystrophic muscles in mice and dogs. In addition, HLA-matched MABs are currently being tested in a phase 1 clinical study on Duchenne MD patients (EudraCT #2011-000176-33). Many reports indicate that the Notch pathway regulates muscle regeneration and satellite cell commitment. However, little is known about Notch-mediated effects on other resident myogenic cells. To possibly potentiate MAB-driven regeneration in vivo, we asked whether Notch signaling played a pivotal role in regulating MAB myogenic capacity. Through different approaches of loss- and gain-of-function in murine and human MABs, we determined that the interplay between Delta-like ligand 1 (Dll1)-activated Notch1 and Mef2C supports MAB commitment in vitro and ameliorates engraftment and functional outcome after intra-arterial delivery in dystrophic mice. Furthermore, using a transgenic mouse model of conditional Dll1 deletion, we demonstrated that Dll1 ablation, either on the injected cells, or on the receiving muscle fibers, impairs MAB regenerative potential. Our data corroborate the perspective of advanced combinations of cell therapy and signaling tuning to enhance therapeutic efficaciousness of somatic stem cells.Notch signaling consists of a conserved pathway, triggered by physical interaction between one ligand and one receptor, both transmembrane proteins exposed by contacting cells.¹ Notch signaling has been involved in different stages of muscle formation² and regeneration.^3,4 The canonical signaling encompasses five ligands (Dll1/3/4, Jagged1/2) and four receptors (Notch1–4); however, the axis Dll1-Notch1 appears consistently involved during myogenic fate specification, for example, neural crest-driven somite maturation.⁵ Moreover, murine embryos expressing a hypomorphic allele of the Notch ligand Dll1 displayed marked impairment of skeletal muscle formation.⁶ Interestingly, the Notch pathway may exert different effects according to the cell context. Culture on DLL1-coated plastic improved ex vivo proliferation and in vivo engraftment of canine satellite cells.⁷ Expression of the active Notch1 intracellular domain (NICD) robustly committed murine and rat mesenchymal stem cells toward the myogenic fate both in vitro and in vivo.⁸ However, Notch-mediated effects on the regenerative potential of non-satellite resident myogenic cells are still unknown.Mesoangioblasts (MABs) are non-satellite resident myogenic stem cells, able to circulate and regenerate dystrophic skeletal muscles.^9,10 HLA-matched MABs are currently under phase 1 clinical study on Duchenne muscular dystrophy patients (EudraCT #2011-000176-33). In this view, understanding the cell-specific effects and mechanisms of myogenic cues will help improving clinical translation of MAB-based therapies in vivo. Recently, it has been shown that Notch synergizes with Pdgf-bb to convert fetal myoblasts into myogenic pericytes.¹¹ However, knowledge about Notch-triggered effects on the regenerative potency of somatic MABs is still scant, particularly in the contexts of cell–cell (in vitro) and fiber–cell (in vivo) contact.Therefore, we asked whether the Dll1-Notch1 axis regulates the myogenic potential of murine and human MABs and how to tune this pathway to ameliorate in vivo MAB-driven regeneration.     

C-terminal fragment of human laminin-binding protein contains a receptor domain for Venezuelan equine encephalitis and tick-borne encephalitis viruses

Polyclonal and monoclonal antibodies (MABs) to human laminin-binding protein (LBP) can efficiently block the penetration of some alphaand flaviviruses into the cell. A panel of 13 types of MABs to human recombinant LBP was used for more detailed study of the mechanism of this process. Competitive analysis has shown that MABs to LBP can be divided into six different competition groups. MABs 4F6 and 8E4 classified under competition groups 3 and 4 can inhibit the replication of Venezuelan equine encephalitis virus (VEEV), which is indicative of their interaction with the receptor domain of LBP providing for binding with virions. According to enzyme immunoassay and immunoblotting data, polyclonal anti-idiotypic antibodies to MABs 4F6 and 8E4 modeling paratopes of the LBP receptor domain can specifically interact with VEEV E2 protein and tick-borne encephalitis virus (TBEV) E protein. Mapping of binding sites of MABs 4F6 and 8E4 with LBP by constructing short deletion fragments of the human LBP molecule has shown that MAB 8E4 interacts with the fragment of amino acid residues 187–210, and MAB 4F6 interacts with the fragment of residues 263–278 of LBP protein, which is represented by two TEDWS peptides separated by four amino acid residues. This suggested that the LBP receptor domain interacting with VEEV E2 and TBEV E viral proteins is located at the C-terminal fragment of the LBP molecule. A model of the spatial structure of the LBP receptor domain distally limited by four linear loops (two of which are represented by experimentally mapped regions of amino acid residues 187–210 and 263–278) as well as the central β-folded region turning into the α-helical site including residues 200–216 of the LBP molecule and providing for the interaction with the laminin-1 molecule has been proposed.     

A comparison of the carcinogen-DNA adducts formed in rat liver in vivo after administration of single or multiple doses of N-methyl-4-aminoazobenzene

N-Methyl-4-aminoazobenzene (MAB) is believed to be metabolized in the liver to an electrophilic N-sulfonyloxy ester which binds covalently to cellular macromolecules, resulting in the induction of hepatic neoplasia. Previous in vivo studies in the rat detected only two hepatic MAB-DNA adducts, 3-(deoxyguanosin-N²-yl)-MAB(N²-dG) and N-(deoxyguanosin-8-yl)-MAB(C8-dG), which respectively accounted for 25% and 70% of the total MAB bound to DNA at 8 h after a single dose of the carcinogen. Subsequently, the C8-dG adduct was shown to be rapidly lost from the DNA while the N²-dG adduct was a persistent lesion. Since a single dose of MAB is not sufficient for complete carcinogenic activity, we sought to identify the MAB-DNA adducts present in rat liver after multiple oral doses of [³H]MAB. The MAB was administered by intubation at a level of 0.2 mmol/kg for 1, 3 or 4 doses and animals were sacrificed at 8 h after the last dose. Hepatic DNA was isolated by extraction and hydroxylapatite chromatography and was enzymatically hydrolyzed to MAB-mononucleoside adducts, which were quantitated by high pressure liquid chromatography (HPLC). After 3 doses, N²-dG, C8-dG, and an unknown adduct were detected. By 4 doses, these accounted for 51%, 25% and 23% of the total adducts. This data is consistent with rapid removal of the C8-dG derivative and the relative persistence of the N²-dG and the unknown adduct. The latter was shown to exhibit chromatographic and pH-dependent solvent partitioning properties that were identical to a product also present in DNA treated with the synthetic ultimate carcinogen, N-benzoyloxy-MAB. Analysis of this adduct by field desorption mass spectrometry (M⁺ = 460) and, after perdeuteromethylation, by electron impact mass spectrometry (M⁺ = 528; M-N(CH₃)(CD₃) = 481) indicated the structure to be a deoxyadenosin-N⁶-yl derivative substituted through an aromatic ring of MAB. Further analysis by 270 MHz ¹H-NMR spectroscopy allowed complete assignment of the MAB and adenyl resonances and was uniquely consistent with a 3-(deoxyadenosin-N⁶-yl)-MAB structure. Since this persistent adduct is potentially mutagenic due to possible tautomeric equilibria between the N⁶-amino and N⁶-imino structures, it may represent an initiating lesion in MAB hepatocarcinogenesis.     

Stratification of Activity and Bacterial Community Structure in Biofilms Grown on Membranes Transferring Oxygen

Previous studies have shown that membrane-aerated biofilm (MAB) reactors can simultaneously remove carbonaceous and nitrogenous pollutants from wastewater in a single reactor. Oxygen is provided to MABs through gas-permeable membranes such that the region nearest the membrane is rich in oxygen but low in organic carbon, whereas the outer region of the biofilm is void of oxygen but rich in organic carbon. In this study, MABs were grown under similar conditions but at two different fluid velocities (2 and 14 cm s⁻¹) across the biofilm. MABs were analyzed for changes in biomass density, respiratory activity, and bacterial community structure as functions of biofilm depth. Biomass density was generally highest near the membrane and declined with distance from the membrane. Respiratory activity exhibited a hump-shaped profile, with the highest activity occurring in the middle of the biofilm. Community analysis by PCR cloning and PCR-denaturing gradient gel electrophoresis of 16S rRNA genes demonstrated substantial stratification of the community structure across the biofilm. Population profiles were also generated by competitive quantitative PCR of gene fragments specific for ammonia-oxidizing bacteria (AOB) (amoA) and denitrifying bacteria (nirK and nirS). At a flow velocity of 14 cm s⁻¹, AOB were found only near the membrane, whereas denitrifying bacteria proliferated in the anoxic outer regions of the biofilm. In contrast, at a flow velocity of 2 cm s⁻¹, AOB were either not detected or detected at a concentration near the detection limit. This study suggests that, under the appropriate conditions, both AOB and denitrifying bacteria can coexist within an MAB.     

Effect of bulk liquid BOD concentration on activity and microbial community structure of a nitrifying,membrane-aerated biofilm

Membrane-aerated biofilms (MABs) are an effective means to achieve nitrification and denitrification of wastewater. In this research, microsensors, fluorescence in situ hybridization (FISH), and modeling were used to assess the impact of bulk liquid biological oxygen demand (BOD) concentrations on the activity and microbial community structure of nitrifying MABs. With 1 g m⁻³ BOD in the bulk liquid, the nitrification rate was 1.3 g N m⁻² day⁻¹, slightly lower than the 1.5 g N m⁻² day⁻¹ reported for no bulk liquid BOD. With bulk liquid BOD concentrations of 3 and 10 g m⁻³, the rates decreased to 1 and 0.4 g N m⁻² day⁻¹, respectively. The percent denitrification increased from 20% to 100% when the BOD increased from 1 to 10 g m⁻³ BOD. FISH results indicated increasing abundance of heterotrophs with increasing bulk liquid BOD, consistent with the increased denitrification rates. Modeling was used to assess the effect of BOD on nitrification rates and to compare an MAB to a conventional biofilm. The model-predicted nitrification rates were consistent with the experimental results. Also, nitrification in the MAB was much less sensitive to BOD inhibition than the conventional biofilm. The MAB achieved concurrent nitrification and denitrification, whereas little denitrification occurred in the conventional biofilm.     

Effects of monoclonal antibodies on α-staphylotoxin action against erythrocytes and model phospholipid membranes

It was found that one of twenty tested monoclonal antibodies (MABs) existed which drastically enhanced ability of Staphylococcus aureus α-tosin (ST) to both lysis of human erythrocytes and increase of planar phospholipid bilayer conductance more than 10 and 1000 times respectively. Other 19 MABs possessed only neutralized effect. The activation could only be observed if the activating MAB (AMAB) interacted with ST in solution but not in membrane. The one molecule of AMAB was able to activate approximately 2–4 molecules of ST. It was assumed that this activation was a result of the AMAB-induced transition of ST from a hydrophilic to an amphiphilic form. The activation could not be observed when the activity of AMAB/ST mixtures was tested on highly sensitive rabbit erythrocytes. All the tested MABs (including AMAB) were able to inhibit the ST-induced lysis of rabbit erythrocytes. The activating effects of AMAB on ST action in BLM and in human erythrocytes as well as their inhibiting influence on the ability of toxin to cause a lysis of rabbit erythrocytes indicate the presence of an ST-specific receptor on the membrane of rabbit erythrocytes.     

Mycobacterium abscessus MAB2560 induces maturation of dendritic cells via Toll-like receptor 4 and drives Th1 immune response

In this study, we showed that Mycobacterium abscessus MAB2560 induces the maturation of dendritic cells (DCs), which are representative antigen-presenting cells (APCs). M. abscessus MAB2560 stimulate the production of pro-inflammatory cytokines [interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β, and IL-12p70] and reduce the endocytic capacity and maturation of DCs. Using TLR4^-/- DCs, we found that MAB2560 mediated DC maturation via Toll-like receptor 4 (TLR4). MAB2560 also activated the MAPK signaling pathway, which was essential for DC maturation. Furthermore, MAB2560-treated DCs induced the transformation of naïve T cells to polarized CD4⁺ and CD8⁺ T cells, which would be crucial for Th1 polarization of the immune response. Taken together, our results indicate that MAB2560 could potentially regulate the host immune response to M. abscessus and may have critical implications for the manipulation of DC functions for developing DC-based immunotherapy. [BMB Reports 2014;47(9): 512-517]     

Reactive oxygen species derived from NAD(P)H oxidase play a role on ethanol-induced hypertension and endothelial dysfunction in rat resistance arteries

Chronic ethanol consumption is a risk factor for cardiovascular diseases. We studied whether NAD(P)H oxidase-derived reactive oxygen species (ROS) play a role in ethanol-induced hypertension, vascular dysfunction, and protein expression in resistance arteries. Male Wistar rats were treated with ethanol (20 %?v/v) for 6 weeks. Ethanol treatment increased blood pressure and decreased acetylcholine-induced relaxation in the rat mesenteric arterial bed (MAB). These responses were attenuated by apocynin (30 mg/kg/day; p.o. gavage). Ethanol consumption increased superoxide anion (O₂ ^?) generation and decreased nitrate/nitrite (NO _x ) concentration in the rat MAB and apocynin prevented these responses. Conversely, ethanol did not affect the concentration of hydrogen peroxide (H₂O₂) and reduced glutathione (GSH) or the activity of superoxide dismutase (SOD) and catalase (CAT) in the rat MAB. Ethanol increased interleukin (IL)-10 levels in the rat MAB but did not affect the levels of tumor necrosis factor (TNF)-α, IL-6, or IL-1β. Ethanol increased the expression of Nox2 and the phosphorylation of SAPK/JNK, but reduced eNOS expression in the rat MAB. Apocynin prevented these responses. However, ethanol treatment did not affect the expression of Nox1, Nox4, p38MAPK, ERK1/2, or SAPK/JNK in the rat MAB. Ethanol increased plasma levels of TBARS, TNF-α, IL-6, IL-1β, and IL-10, whereas it decreased NO _x levels. The major finding of our study is that NAD(P)H oxidase-derived ROS play a role on ethanol-induced hypertension and endothelial dysfunction in resistance arteries. Moreover, ethanol consumption affects the expression and phosphorylation of proteins that regulate vascular function and NAD(P)H oxidase-derived ROS play a role in such responses.     

Monoclonal antibodies to the collagen binding domain of human plasma fibronectin

Five independent hybrids producing monoclonal antibodies to human plasma fibronectin have been obtained by fusing P3/X63-Ag8 myeloma cells with immune mouse splenocytes. The specificity of these monoclonal antibodies (MABs) for fibronectin was demonstrated by three independent tests: binding to the purified soluble molecule, immunofluorescence staining of insoluble extracellular matrices produced by endothelial cells in vitro, immunostaining of fibronectin tryptic peptides after separation on SDS-PAGE and transfer to nitrocellulose sheets. Two antibodies (MAB 29 and 52) recognized selectively human fibronectin while the others (MAB 5, 30 and 59) reacted also with plasma fibronectin from calf, hamster and chicken. Four distinct epitopes were recognized by the MABs studied. MAB 5, 30, 52 and 59 reacted with distinct antigenic sites, while MAB 29 and 52 bind to the same site. Antigenic fragments were identified by immunostaining of fibronectin tryptic peptides. MAB 5 reacted with a collagen binding fragment with a molecular weight of 120 K. In addition, each of the MAB 29, 30, 52 and 59 reacted with peptides with a molecular weight of 40 K that bind to gelatin. Since these antibodies do not inhibit fibronectin-collagen interaction, it is concluded that their corresponding epitopes are clustered in a region close, but not coincident, to the collagen binding site of fibronectin.     

Use of monoclonal antibodies for identification of growth-controlling neuropeptides in the mussel Mytilus edulis (Mollusca: Bivalvia)

Monoclonal antibodies were developed against cerebral ganglia (CG) of the mussel Mytilus edulis by the immunization of mice with unpurified homogenates of these organs. The screening protocol of hybridoma was based upon immunohistological observations of cytocentrifugated ganglia cells. A panel of 29 monoclonal antibodies (MABs) specific of CG epitopes was harvested and subsequently used for the immunocytochemical study of CG cells. Several subpopulations of ganglia cells were specifically revealed by MABs. Identification of epitopes involved in growth control was approached via the application of a bioassay allowing the assessment of protein synthesis stimulation. MAB 42 and 46 affected amino acid incorporation induced by CG extract. These results lead to the conclusion that the epitopes recognised by these antibodies are involved in growth control. An immunoenzymatic assay was performed with CG extracts for quantitative analyses of epitopes.     

Antibody-assisted enhancement of biological activities of CXCL14 in human monocytic leukemia-derived THP-1 cells and high fat diet-induced obese mice

CXCL14 is a CXC-type chemokine acting on tissue macrophages, immature dendritic cells, natural killer cells, and epithelial tumor cells. It also serves as a metabolic regulator in obese mice by blunting insulin activity. In contrast to other CXC chemokines, it remains to be clarified how CXCL14 activates its putative receptors on the cell surface and whether it induces chemokinesis. This is mainly due to the insufficient sensitivity of currently available bioassays for CXCL14. In this study, we found that the anti-CXCL14 monoclonal antibody, MAB730, remarkably enhances the activities of CXCL14 in human monocytic leukemia-derived THP-1 cells and immature dendritic cells. MAB730 augmented CXCL14-mediated chemotaxis and chemokinesis with distinct dose requirement. Chemotaxis inducing activity was retained in the MAB730 F(ab')₂ fraction, but not in the Fab fraction, implying that ligand dimerization is involved in the MAB730-assisted enhancement of CXCL14 activity. In addition, MAB730 was more efficient than heparin at inhibiting CXCL14 binding to low affinity receptors on THP-1 cells. Finally, in vivo administration of MAB730 antibody into high fat diet-induced obese mice increased whole body insulin resistance and glucose intolerance. These unique properties of MAB730 will be useful for elucidating the molecular mechanism of cellular responses elicited by CXCL14.     

Vaccinia Viral Protein A27 Is Anchored to the Viral Membrane via a Cooperative Interaction with Viral Membrane Protein A17

The vaccinia viral protein A27 in mature viruses specifically interacts with heparan sulfate for cell surface attachment. In addition, A27 associates with the viral membrane protein A17 to anchor to the viral membrane; however, the specific interaction between A27 and A17 remains largely unclear. To uncover the active binding sites and the underlying binding mechanism, we expressed and purified the N-terminal (18–50 residues) and C-terminal (162–203 residues) fragments of A17, which are denoted A17-N and A17-C. Through surface plasmon resonance, the binding affinity of A27/A17-N (K_A = 3.40 × 10⁸ m⁻¹) was determined to be approximately 3 orders of magnitude stronger than that of A27/A17-C (K_A = 3.40 × 10⁵ m⁻¹), indicating that A27 prefers to interact with A17-N rather than A17-C. Despite the disordered nature of A17-N, the A27-A17 interaction is mediated by a specific and cooperative binding mechanism that includes two active binding sites, namely ³²SFMPK³⁶ (denoted as F1 binding) and ²⁰LDKDLFTEEQ²⁹ (F2). Further analysis showed that F1 has stronger binding affinity and is more resistant to acidic conditions than is F2. Furthermore, A27 mutant proteins that retained partial activity to interact with the F1 and F2 sites of the A17 protein were packaged into mature virus particles at a reduced level, demonstrating that the F1/F2 interaction plays a critical role in vivo. Using these results in combination with site-directed mutagenesis data, we established a computer model to explain the specific A27-A17 binding mechanism.     

<Emphasis Type="Italic">Deinococcus sedimenti</Emphasis> sp. nov. isolated from river sediment

A novel Gram-positive, oval-shaped, non-motile bacterium designated strain 16F1L^T was isolated from sediment collected from the Han River in Seoul, Republic of Korea. Based on the 16S rRNA gene sequence (1,448 bp), this strain was identified as a member of the genus Deinococcus that belongs to the class Deinococci. Similarities in the 16S rRNA gene sequence were shown with Deinococcus daejeonensis MJ27^T (99.0%), D. grandis DSM 3963^T (98.1%), D. radiotolerans C1^T (97.5%), and D. caeni Ho-08^T (97.2%). Strain 16F1L^T was classified as a different genomic species from closely related Deinococcus members, based on less than 70% DNA-DNA relatedness. Genomic DNA G+C content of strain 16F1L^T was 67.2 mol%. Strain 16F1L^T was found to grow at temperatures of 10–37°C (optimum 25°C) and pH 7–8 (optimum pH 7) on R2A medium, and was catalase-positive and oxidase-negative. Strain 16F1L^T showed resistance to gamma radiation (D₁₀ > 2 kGy). In addition, this strain had the following chemotaxonomic characteristics: the major fatty acids were C_15:1ω6c and C_16:1ω7c; the polar lipid profile contained phosphoglycolipids, unknown aminophospholipids, an unknown aminoglycolipid, unknown aminolipids, an unknown glycolipid, an unknown phospholipid, and an unknown polar lipid; the major quinone was MK-8. Phylogenetic, genotypic, phenotypic, and chemotaxonomic characteristics indicated that strain 16F1L^T represents a novel species within the genus Deinococcus, for which the name Deinococcus sedimenti sp. nov. is proposed. The type strain is 16F1L^T (=KCTC 33796^T =JCM 31405^T).     

Interactions of human 8-oxoguanine DNA glycosylase with single-and double-stranded DNAs

The interaction of human 8-oxoguanine (8-oxoG) DNA glycosylase (hOGG1) with single-and double-stranded oligodeoxyribonucleotides (ODNs) was studied by a method of stepwise increase in ligand complexity. ODNs were shown to act as competitive inhibitors with respect to the substrate of the reaction catalyzed by hOGG1. K _I was estimated for various homo-and hetero-ODNs. All nucleotides covered by the enzyme globule proved to additively interact with hOGG1. An increase in the ODN size n by one nucleotide or base pair in d(pN)_n and their duplexes monotonically increased their affinity for hOGG1 by a factor of 1.4–1.5 until n = 10, mostly due to weak nonspecific additive contacts between hOGG1 and the sugar-phosphate backbone. Weak nonspecific additive interactions contributed about five orders of magnitude to the total affinity of hOGG1 for specific DNA (K _d ～ 10^?5 M). Specific 8-oxoG increased the affinity of DNA for the enzyme by three orders of magnitude (K _d ～ 10^?8 M). The main features of the recognition of specific DNA by hOGG1 were analyzed.     

Production and characterization of a panel of monoclonal antibodies to Toxoplasma gondii antigens

A representative collection was obtained containing 68 monoclonal antibodies (MAB) to Toxoplasma gondii antigens, which was characterized by the binding with the below fractions of tochizoites in the immune-enzyme assay (IEA) and immunoblotting (IB): membrane (MEM), somatic (water-soluble, SOM) and excretory-secretory (ES). Most of MABs were produced to MEM antigens (43), 6 MABs reacted with the somatic fraction, and 3 MABs reacted with both fractions. Two MABs to ES antigen were detected in the latter group. An analysis of MABs in concurrent IEA and IB revealed the immune-dominant proteins of the MEM and SOM fractions of antibodies to T. gondii tochizoites (p30 and p27, respectively). The presence of 2 non-overlapping antigenic determinants was shown for p30. Further research would detect MABs that could be used in the diagnosis of toxoplasmosis.     

Antirestriction activity of the monomeric and dimeric forms of T7 Ocr

The Ocr antirestriction protein, which is encoded by bacteriophage T7 0.3 (ocr), specifically inhibits type I restriction-modification enzymes. Ocr belongs to a family of DNA-mimicking proteins. Native Ocr forms homodimers both in solution and in crystal. Ocr mutants with two amino acid substitutions (Orc F53D A57E and Ocr F53R V77D) were constructed to occur as monomers in solution. The dissociation constant K _d for the Ocr complex with EcoKI (R₂M₂S) proved to differ by three orders of magnitude between the (Ocr)₂ dimer and Ocr F53D A57E and Ocr F53R V77D monomers (10^?10 M vs. 10^?7 M). Antimodification activity was substantially lower in the Ocr monomers. The dimeric form found to be essential for high inhibitory activity of Ocr.     

Fluorine-19 magnetic resonance angiography of the mouse

Purpose

To implement and characterize a fluorine-19 (¹⁹F) magnetic resonance imaging (MRI) technique and to test the hypothesis that the ¹⁹F MRI signal in steady state after intravenous injection of a perfluoro-15-crown-5 ether (PCE) emulsion may be exploited for angiography in a pre-clinical in vivo animal study.

Materials and Methods

In vitro at 9.4T, the detection limit of the PCE emulsion at a scan time of 10 min/slice was determined, after which the T₁ and T₂ of PCE in venous blood were measured. Permission from the local animal use committee was obtained for all animal experiments. 12 µl/g of PCE emulsion was intravenously injected in 11 mice. Gradient echo ¹H and ¹⁹F images were obtained at identical anatomical levels. Signal-to-noise (SNR) and contrast-to-noise (CNR) ratios were determined for 33 vessels in both the ¹⁹F and ¹H images, which was followed by vessel tracking to determine the vessel conspicuity for both modalities.

Results

In vitro, the detection limit was ∼400 µM, while the ¹⁹F T₁ and T₂ were 1350±40 and 25±2 ms. The ¹⁹F MR angiograms selectively visualized the vasculature (and the liver parenchyma over time) while precisely coregistering with the ¹H images. Due to the lower SNR of ¹⁹F compared to ¹H (17±8 vs. 83±49, p<0.001), the ¹⁹F CNR was also lower at 15±8 vs. 52±35 (p<0.001). Vessel tracking demonstrated a significantly higher vessel sharpness in the ¹⁹F images (66±11 vs. 56±12, p = 0.002).

Conclusion

¹⁹F magnetic resonance angiography of intravenously administered perfluorocarbon emulsions is feasible for a selective and exclusive visualization of the vasculature in vivo.