The C-terminal domain of Escherichia coli ribosomal protein L7/L12 can occupy a location near the factor-binding domain of the 50S subunit as shown by cross-linking with N-[4-(p-azidosalicylamido)butyl]-3-(2'-pyridyldithio)propionamide.

All large ribosomal subunits contain two dimers composed of small acidic proteins that are involved in binding elongation factors during protein synthesis. The ribosomal location of the C-terminal globular domain of the Escherichia coli ribosomal acidic protein L7/L12 has been determined by protein cross-linking with a new heterobifunctional, reversible, photoactivatable reagent, N-[4-(p-azidosalicylamido)-butyl]-3-(2'-pyridyldithio)propionamide . Properties of this reagent are described. It was first radiolabeled with 125I and then attached through the formation of a disulfide bond to a unique cysteine of L7/L12, introduced by site-directed mutagenesis at residue 89. Intact 50S ribosomal subunits were reconstituted from L7/L12-depleted cores and the radiolabeled L7/L12Cys89. Irradiation of the reconstituted subunits resulted in photo-cross-linking between residue 89 and other ribosomal components. Reductive cleavage of the disulfide cross-link resulted in transfer of the 125I label from L7/L12Cys89 to the other cross-linked components. Two radiolabeled proteins were identified, L11 and L10. The location of both of these proteins is well established to be at the base of the L7/L12 stalk near the binding sites for the N-terminal domain of both L7/L12 dimers, and for elongation factors. The result indicates that L7/L12 can have a bent conformation bringing the C-terminal domain of at least one of the L7/L12 dimers at or near the factor-binding domain. The cross-linking method with radiolabeled N-[4-(p-azidosalicylamido)butyl]-3-(2'-pyridyldithio)propionamide should be applicable for studies of other multicomponent complexes that can be reconstituted.     

Interaction of Topotecan,DNA Topoisomerase I Inhibitor,with Double-Stranded Polydeoxyribonucleotides. 1. Topotecan Dimerization in Solution

Behavior of topotecan, DNA topoisomerase I inhibitor, was studied in aqueous solutions by optical methods. Topotecan absorption spectra were recorded in the pH range 0.5–11.5 and its pKa were determined. Quantum chemical calculations were made for all charge states of the topotecan molecule in lactone and carboxylate form. The calculated absorption maxima agree well with the experimental data. Protonation of the topotecan D ring (pKa 3.6) was revealed. Comparison of experimental and calculated data showed topotecan structure with a proton at the oxygen atom at C16a rather than N4 to be the most preferable. Topotecan molecules were shown to form dimers at concentrations above 10^–5M. Topotecan dimerization is accompanied by an increase in the pKa of hydroxy group of the A ring from 6.5 ([TPT] = 10^–6M) to 7.1 ([TPT] = 10^–4M), which indicates participation of this group in dimer stabilization, perhaps due to intermolecular hydrogen bonding with N1 of the B ring of a neighboring molecule. Probable dimer structures were proposed. The topotecan dimerization constant was determined, K = (4.0 ± 0.7)·10³M^–1.     

Immunoassays based on electrochemical detection using microelectrode arrays

We show that CombiMatrix's VLSI arrays of individually addressable electrodes, using conventional CMOS integrated circuitry, can be used in detecting various analytes via immunoassay protocols. These microarrays provide over 1000 electrodes per square centimeter. The chips are coated with a porous material on which specific affinity tags are synthesized proximate to selected electrode sites. CombiMatrix microarrays are used to develop spatially multiplexed assay formats for biological entities over a wide range of sizes, from small molecules to cells. Antibodies are tagged with coded affinity labels and then allowed to self-assemble on the appropriate electrode assay sites. Each analyte-specific antibody is chaperoned to individual, predetermined locations by the self-assembly process. The resulting chip can perform numerous different analyte-specific immunoassays, simultaneously. We present new detection technologies based upon the use of the active individually addressable microelectrodes on the chip: redox enzyme amplified electrochemical detection. The results for human alpha1 acid glycoprotein, ricin, M13 phage, Bacillus globigii spores, and fluorescein indicate that this method is one of the most sensitive available, with limits of detection in the attomole range. The detection range is 4-5 logs of analyte concentration, with an assay volume of 50 microl or less. The system provides for a host of multiplexed immunoassays because of the large number of electrodes available. We show how the assays can be optimized for maximum performance on the CombiMatrix microarray platform.     

Self-assembling protein arrays using electronic semiconductor microchips and in vitro translation

Protein arrays will greatly accelerate research and development in medical and biological sciences. We have used cell-free protein biosynthesis and a parallel immobilization strategy for producing protein biochips. We demonstrate a model two-protein microarray using luciferase and green fluorescent protein, both expressed in a cell-free system and specifically immobilized on CombiMatrix semiconductor oligonucleotide microarrays. This demonstration provides evidence for the appropriate folding, activity, robust presentation, and efficient flexible detection of proteins on the microscale.     

Raman and surface-enhanced Raman scattering spectroscopy of bis-netropsins and their DNA complexes

The interactions of three bis-netropsins (bis-Nts), which are potent catalytic inhibitors of DNA-binding enzymes, with three double-stranded oligonucleotides (OLIGs), which contain sites of different specific affinities for each bis-Nt, were analyzed. Raman spectroscopy was performed for selective monitoring of modifications of the bis-Nt or the OLIG structure upon bis-Nt-DNA binding, and surface-enhanced Raman scattering spectroscopy (SERS) was an additional tool for topology studies of ligand-DNA complexes. The spectral data showed conformational changes of both partners (bis-Nt and OLIG) upon complexation. Structural variations of bis-Nts appeared to be dependent on a bis-Nt-OLIG binding constant and were found to be small in the specific DNA binding and highest for nonspecific binding of bis-Nt with the corresponding OLIG. The conformational changes of the OLIGs were varied with a bis-Nt-OLIG binding constant in the same manner. The bis-Nts seemed to induce a perturbation in the OLIG's structure, as well as in the positions of their direct binding. These DNA structural modification effects may explain the inhibition of DNA-binding enzymes in the variety of very distinct DNA-enzyme binding sites by bis-Nts reported previously.     

Malaria Parasite Plasmodium falciparum Proteins on the Surface of Infected Erythrocytes as Targets for Novel Drug Discovery

Biochemistry (Moscow) - Specific adhesion (sequestration) of Plasmodium falciparum parasite-infected erythrocytes (IEs) in deep vascular beds can cause severe complications resulting in death. This...     

A novel mtDNA ND6 gene mutation associated with LHON in a Caucasian family

Leber's hereditary optic neuropathy (LHON) is a frequent cause of inherited blindness. A routine screening for common mtDNA mutations constitutes an important first in its diagnosis. However, a substantial number of LHON patients do not harbor known variants, both pointing to the genetic heterogeneity of LHON and bringing into question its genetic diagnosis. We report a familial case that exhibited typical features of LHON but lacked any of the common mutations. Genetic analysis revealed a novel pathogenic defect in the ND6 gene at 14279A that was not detected in any haplogroup-matched controls screened for it, nor has it been previously reported. This mutation causes a substantial conformational change in the secondary structure of the polypeptide matrix coil and may explain the LHON expression. Thus, it expands the spectrum of deleterious changes affecting ND6-encoding subunit and further highlights the functional significance of this gene, providing additional clues to the disease pathogenesis.     

Biocompatible fluorescent nanocrystals for immunolabeling of membrane proteins and cells

A methodology for simple convenient preparation of bright, negatively or positively charged, water-soluble CdSe/ZnS core/shell nanocrystals (NCs) and their stabilization in aqueous solution is described. Single NCs can be detected using a standard epifluorescent microscope, ensuring a detection limit of one molecule coupled with an NC. NCs solubilized in water by DL-Cys were stabilized, to avoid aggregation, by poly(allylamine) and conjugated with polyclonal anti-mouse antibodies (Abs). NC-Abs conjugates were tested in dot-blots and exhibited retention of binding capacity within several nanograms of antigen detected. We further demonstrated the advantages of NC-Abs conjugates in the immunofluorescent detection and three-dimensional (3D) confocal analysis of p-glycoprotein (p-gp), one of the main mediators of the MDR phenotype, overexpressed in the membrane of MCF7r breast adenocarcinoma cells. Immunolabeling of p-gp with NC-Abs conjugates was 4200-, 2600-, and 420-fold more resistant to photobleaching than its labeling with fluorescein isothiocyanate-Abs, R-phycoerythrin-Abs, and AlexaFluor488-Abs, respectively. The labeling of p-gp with NC-Abs conjugates was highly specific, and the data were used for confocal reconstruction of 3D images of the p-gp distribution in the MCF7r cell membrane. Finally, we demonstrated the applicability of NC-Abs conjugates obtained by the method described to specific detection of antigens in paraffin-embedded formaldehyde-fixed cancer tissue specimens, using immunostaining of cytokeratin in skin basal carcinoma as an example. We conclude that the NC-Abs conjugates may serve as easy-to-do, highly sensitive, photostable labels for immunofluorescent analysis, immunohistochemical detection, and 3D confocal studies of membrane proteins and cells.     

A plasma survey using 38 PfEMP1 domains reveals frequent recognition of the Plasmodium falciparum antigen VAR2CSA among young Tanzanian children

PfEMP1 proteins comprise a family of variant antigens that appear on the surface of P. falciparum-infected erythrocytes and bind to multiple host receptors. Using a mammalian expression system and BioPlex technology, we developed an array of 24 protein constructs representing 38 PfEMP1 domains for high throughput analyses of receptor binding as well as total and functional antibody responses. We analyzed the reactivity of 561 plasma samples from 378 young Tanzanian children followed up to maximum 192 weeks of life in a longitudinal birth cohort. Surprisingly, reactivity to the DBL5 domain of VAR2CSA, a pregnancy malaria vaccine candidate, was most common, and the prevalence of reactivity was stable throughout early childhood. Reactivity to all other PfEMP1 constructs increased with age. Antibodies to the DBL2βC2(PF11_0521) domain, measured as plasma reactivity or plasma inhibition of ICAM1 binding, predicted reduced risk of hospitalization for severe or moderately severe malaria. These data suggest a role for VAR2CSA in childhood malaria and implicate DBL2βC2(PF11_0521) in protective immunity.