Site IIA Research Articles

Interaction between holo transferrin and HSA–PPIX complex in the presence of lomefloxacin: An evaluation of PPIX aggregation in protein–protein interactions

Human serum albumin (HSA) and holo transferrin (TF) are two serum carrier proteins that are able to interact with each other, thereby altering their binding behavior toward their ligands. During the course of this study, the interaction between HSA–PPIX and TF, in the presence and absence of lomefloxacin (LMF), was for the first time investigated using different spectroscopic and molecular modeling techniques. Fluorescence spectroscopy experiments were performed in order to study conformational changes of proteins. The RLS technique was utilized to investigate the effect of LMF on J-aggregation of PPIX, which is the first report of its kind. Our findings present clear-cut evidence for the alteration of interactions between HSA and TF in the presence of PPIX and changes in drug-binding to HSA and HSA–PPIX complex upon interaction with TF. Moreover, molecular modeling studies suggested that the binding site for LMF became switched in the presence of PPIX, and that LMF bound to the site IIA of HSA. The obtained results should give new insight into research in this field and may cast some light on the dynamics of drugs in biological systems.

Binding of a bcl-2 Family Inhibitor to Bovine Serum Albumin: Fluorescence Quenching and Molecular Docking Study

Both fluorescence spectroscopic and molecular docking methods were used to investigate the interaction between bovine serum albumin (BSA) and a known Bcl-xl/Bcl-2 inhibitor HA 14-1. Based on the spectral overlap between the emission of BSA and absorption of HA 14-1, Forster energy transfer was proposed to be the possible quenching mechanism. The Stern-Volmer constants are 2.49 x 10⁴, 2.04 x 10⁴ and 0.90 x 10⁴ M⁻¹ at 293, 303 and 318 K, respectively, indicating that a static quenching process dominates. Thermodynamic parameters were further obtained. The derived negative Δ H (-27.51 kJ mol⁻¹) and Δ S (-11.11 J mol⁻¹ K⁻¹) values suggest hydrogen bond interaction and van der Waals force are the main binding force. The docking study was performed on BSA model. According to the docking score and the number of hydrogen bonds, the potential binding site for HA 14-1 is proposed to be the site IIA, also known as drug site 1.

Successional changes in ectomycorrhizal fungi associated with the polar willow Salix polaris in a deglaciated area in the High Arctic, Svalbard

Polar willow (Salix polaris Wahlenb.), a mycorrhizal dwarf shrub, colonizes recently deglaciated areas in the High Arctic, Svalbard. To clarify successional changes in ECM fungi associated with S. polaris after glacier retreat, we examined the diversity and density of ECM fungi in culture and field conditions. Plant and soil samples were collected from three sites of different successional stages in the deglaciated area of Austre Broggerbreen, near Ny-Alesund, Svalbard. The successional stages were early stage with newly exposed bare ground (site I), transient stage with scattered colonization of Salix (sites IIa and IIb), and late stage with well-developed vegetation (site III). No ECM colonization on Salix was observed in soils collected from bare ground in early and transient stages (sites I and IIa). However, most Salix individuals showed ECM colonization in soils collected from sites close to Salix colonies in transient and late stages (sites IIb and III). Based on molecular analyses and operational taxonomic unit (OTU: >95% ITS sequence similarity) delimitations, we identified 15 OTUs/species in eight genera. The dominant OTU/species of ECM fungi identified in the transient and late stages was Geopora sp.1 and Cenococcum sp.1, respectively. In the culture experiment, ECM diversity was greater in late stage (eight OTUs/species) than in transient stage (three OTUs/species). This pattern was consistent with field observations, i.e., late-stage sites contained more OTUs/species of ECM fungi. These results indicate that species diversity of ECM fungi increases and the dominant species changes with the progress of succession after glacier retreat in the High Arctic.

Development of combined vaccines for rabies and immunocontraception

Rabies prevention and appropriate population management of free-ranging animals have an important role to play in the eventual elimination of rabies in dogs. An effective sterilant based on rabies vaccines has the potential to create a supportive measure of public acceptability and to reduce associated clinic visit costs. We inserted the coding sequence of gonadotropin-releasing hormone (GnRH) into different locations within the rabies virus ERA glycoprotein (G) gene, and demonstrated that the amino terminus (N), antigenic site IIa, and the junction between the ecto- and cytoplasmic domains (C) of the G were suitable sites for GnRH insertion. The rescued recombinant rabies viruses ERA-N-GnRH and ERA-C-GnRH grew as well as the parental ERA virus, reaching 1×109ffu/ml in cell culture. Insertion and expression of the GnRH were stable in the viruses after multiple passages in vitro. To increase immunogenicity of the GnRH peptide, two copies of GnRH, aligned in tandem, were fused to the N terminus of the G. The recombinant rabies virus ERA-N-2GnRH was recovered and grown to high titers in cell culture. All GnRH-carrying rabies viruses induced antibodies against GnRH in immunized mice and protected 100% of the animals after rabies virus challenge. The recombinant viruses reacted strongly with the serum from a GonaCon™-immunized animal. The GnRH-carrying rabies viruses have significant potential in rabies and animal population control.

Elucidation of the human serum albumin (HSA) binding site for the Cu-PTSM and Cu-ATSM radiopharmaceuticals

The Cu-PTSM (pyruvaldehyde bis(N(4)-methylthiosemicarbazonato)copper(II)) and Cu-ATSM (diacetyl bis(N(4)-methylthiosemicarbazonato)copper(II)) radiopharmaceuticals exhibit strong, species-dependent binding to human serum albumin (HSA), while Cu-ETS (ethylglyoxal bis(thiosemicarbazonato)copper(II)) appears to only exhibit nonspecific binding to human and animal serum albumins. This study examines the structural basis for HSA binding of Cu-PTSM and Cu-ATSM via competition with drugs having known albumin binding sites. Warfarin, furosemide, ibuprofen, phenylbutazone, benzylpenicillin, and cephmandole were added to HSA solutions at drug:HSA mole ratios from 0 to 8:1, followed by quantification of radiopharmaceutical binding to HSA by ultrafiltration. Warfarin, a site IIA drug, progressively displaced both [(64)Cu]Cu-PTSM and [(64)Cu]Cu-ATSM from HSA. At 8:1 warfarin:HSA mole ratios, free [(64)Cu]Cu-PTSM and [(64)Cu]Cu-ATSM levels increased 300-500%. This was in contrast to solutions containing ibuprofen, a site IIIA drug; no increase in free [(64)Cu]Cu-PTSM or [(64)Cu]Cu-ATSM was observed except at high ibuprofen:HSA ratios, where secondary ibuprofen binding to the IIA site may cause modest radiopharmaceutical displacement. By contrast, and consistent with earlier findings suggesting Cu-ETS exhibits only nonspecific associations, [(64)Cu]Cu-ETS binding to HSA was unaffected by the addition of drugs that bind in either site. We conclude that the species-dependence of Cu-PTSM and Cu-ATSM albumin binding arises from interaction(s) with the IIA site of HSA.

Settlement of the Early Linear Ceramics Culture at Brunn am Gebirge, Wolfholz site

The early Neolithic sites I to V from Brunn Wolfholz, south of Vienna, were excavated between 1989 and 2005. Till now about 75 longhouses, with the standardized size of 20x7 m have been recognized. The whole settlement has a length from North to South of 850 m and from East to West of 500 m. The oldest part is site IIa in the south; it contains rough ceramics with plastic ornaments, which seem to be in a Starčevo tradition. The Early Linear Ceramics starts only in site III. The youngest phase of the settlement is in the North in site I, which apparently leads already to the Notenkopf Ceramics.

SAR by MS: Discovery of a New Class of RNA-Binding Small Molecules for the Hepatitis C Virus: Internal Ribosome Entry Site IIA Subdomain

A new class of small molecules that bind the HCV RNA IRES IIA subdomain with sub-micromolar affinity is reported. The benzimidazole 'hit' 1 with a KD approximately 100 microM to a 29-mer RNA model of Domain IIA was identified from a 180000-member library using mass spectrometry-based screening methods. Further MS-assisted SAR (structure-activity relationships) studies afforded benzimidazole derivatives with sub-micromolar binding affinity for the IIA RNA construct. The optimized benzimidazoles demonstrated activity in a cellular replicon assay at concentrations comparable to their KD for the RNA target.

PCF1 and PCF2 specifically bind to cis elements in the rice proliferating cell nuclear antigen gene.

We have previously defined the promoter elements, sites IIa and IIb, in the rice proliferating cell nuclear antigen (PCNA) gene that are essential for meristematic tissue-specific expression. In this study, we isolated and characterized cDNAs encoding proteins that specifically bind to sites IIa and IIb. The two DNA binding proteins, designated PCF1 and PCF2, share > 70% homology in common conserved sequences at the N-terminal regions. The conserved regions are responsible for DNA binding and homodimer and heterodimer formation, and they contain a putative basic helix-loop-helix (bHLH) motif. The structure and DNA binding specificity of the bHLH motif are distinguishable from those of other known bHLH proteins that bind to the E-box. The motif is > 70% homologous to several expressed sequence tags from Arabidopsis and rice, suggesting that PCF1 and PCF2 are members of a novel family of proteins that are conserved in monocotyledons and dicotyledons. A supershift assay using an anti-PCF2 antibody showed the involvement of PCF2 in site IIa (site IIb) binding activities in rice nuclear extracts, particularly in meristematic tissues. PCF1 and PCF2 were also more likely to form heterodimers than homodimers. Our results suggest that PCF1 and PCF2 are involved in meristematic tissue-specific expression of the rice PCNA gene through binding to sites IIa and IIb and formation of homodimers or heterodimers.

Two of three promoter elements identified in a rice gene for proliferating cell nuclear antigen are essential for meristematic tissue‐specific expression

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta, and it is highly conserved among eukaryotes. The rice PCNA promoter, truncated to position -263, was previously shown to confer meristematic tissue-specific expression in transgenic plants. By DNasel footprinting and gel retardation analysis, three cis-acting elements were defined in this truncated promoter, which designated site I (-201 to -194, CCAGGTGG), site IIa (-197 to -188, TGGGCCCGT) and site IIb (-178 to -169, TGGTCCCAC). Site I resembles the G-box, and sites IIa and IIb resemble the conserved motif (T/GGTCCCAT) that is found in promoter regions of auxin-regulated genes. Functional analysis of expression of a PCNA-GUS gene fusion in transgenic tobacco plants revealed that a mutation in site I in the full-length 2.0 kb promoter had no significant effect on the activity. However, the mutation in site I in the truncated -263 promoter, which had 14% of the activity of the full-length promoter, caused a considerable decrease in the activity, suggesting that site I contributes in part to transcriptional activation. Simultaneous disruption of sites IIa and IIb in the full-length promoter caused about 80-85% loss of promoter activity, while separate disruption of site IIa or site IIb resulted in no marked change on the activity. These observations suggest that site IIa and site IIb play an important role in the meristematic tissue-specific expression of the rice PCNA gene, presumably by mediating putative enhancer activities dependent on the far-upstream region.

Protective activity of a murine monoclonal antibody against European bat lyssavirus 1 (EBL1) infection in mice

A mouse model was designed to test in vivo the efficacy of rabies immune globulins and specific neutralizing monoclonal antibodies to prevent European bat lyssavirus I infection. Human or equine rabies immune globulins previously found to contain variable amounts of neutralizing bat lyssavirus crossreactive antibodies were passively transferred to mice receiving intramuscularly a lethal dose of bat lyssavirus type 1. Immune globulins did not protect mice well against bat lyssavirus 1 whereas they reduced the mortality caused by rabies virus. In contrast, mice inoculated with bat lyssavirus 1 or rabies virus survived when passively immunized with bat lyssavirus 1 specific monoclonal antibody (mAb 8-2). This monoclonal antibody, an IgG 2a, recognized an epitope located in the antigenic site IIa of rabies glycoprotein. A mutation replacing the lysine 198 by glutamate in a rabies variant abrogated sensitivity to this neutralizing antibody. Because of its broad neutralizing spectrum against wild virus isolates, including European bat lyssaviruses, this monoclonal antibody should be a good candidate for rabies immune globulin replacement. It could improve efficacy of rabies vaccination, used either alone or in conjunction with human rabies immune globulins or monoclonal antibody cocktail to supplement their lack of crossreactivity to European bat lyssavirus 1.

Interaction of molecules with nucleic acids. II. Two pairs of families of intercalation sites, unwinding angles, and the neighbor-exclusion principle.

AbstractIntercalation‐site geometries are generated for a tetramer duplex extracted from B‐DNA. Glycosidic angles and puckers of the deoxyribose sugar groups bonded to base pairs BP1 and BP4, namely, those at either end of the tetramer duplex, are assumed to be those of B‐DNA to insure continuity. All possible geometrical conformations for combinations of C(2′)‐endo, C(3′)‐endo, C(2′)‐exo, and C(3′)‐exo sugar puckers are determined for the tetranucleotide backbone. Those with minimum energy are selected as candidates for intercalation sites. Calculations reveal two pairs of physically meaningful families of intercalation sites which occur in two distinct regions, I and II, of helical angles which orient BP2 relative to BP3 and with the helical axis disjointed between these base pairs. For each site I and II within BP2 and BP3, there are two distinct backbone conformations, A and B, connecting BP3 to BP4 or BP1 to BP2 which do not disrupt backbone conformations connecting BP2 to BP3. Hence two pairs, IA and IB, and IIA and IIB, of intercalation sites exist in which the sugar puckers along the backbone of the tetramer alternate from C(2′)‐endo to C(3′)‐endo on the backbone (5′p3′) connecting BP2 to BP3. The glycosidic angles of the C(3′)‐endo sugar χ3γ are, coincidentally, 80° ± 2° for both conformations γ = A and B connecting BP3 to BP4 along the phosphate backbone (5′p3′). Consistent with the theoretical results, the experimental unwinding angles can be grouped into two categories with absolute values of 18° and 26°. The theoretical unwinding angles for sites IA and IB of 16° and for sites IIA and IIB of 20° occur for a displacement of ‐0.8 Å in the helical axes of BP2 and BP3 and for a 100% G·C composition, with a decrease depending on the amount of A·T base pairs present. Ratios of theoretical unwinding angles of sites I and II, which range from 0.75 to 0.84 for the two principal sites, compare well with the experimental value of 0.71. The theoretical results, in agreement with experimental observation, provide a new interpretation of the nature and conformation of the possible binding sites. Conformations obtained from these studies of intercalation sites in a tetramer duplex are used to rationalize the well‐known neighbor‐exclusion principle. The possibility of violation of this principle is demonstrated by the existence of two families of physically meaningful conformations. Conformations of unconstrained dimer duplexes are also obtained, one of which corresponds to the experimental crystal structure of ethidium–dinucleoside complexes, but these cannot be joined to the B‐DNA structure. Backbone conformations of the tetramer duplex can be constructed until the base‐pair separation reaches 8.25 Å, which may limit the molecules that can intercalate.