Covalent attachment of gold nanoparticles to DNA templates

Functionalized gold nanoparticles have been covalently bound to internal, modified sites on double-stranded DNA. Gold nanoparticles coated with mercaptosuccinic acid or thioctic acid were bound to amino-modified thymine bases on double-stranded DNA. Visible absorption spectra, gel electrophoresis, and atomic force microscopy were used to analyze the products. Thiol groups were added to one end of the gold/nanoparticle product, which was then attached to a gold surface. This method has the potential to allow controlled placement of particles with subnanometer precision and to allow attachment of the product to fixed contacts for nanodevice fabrication.     

Imaging size-selective permeation through micropatterned thin films using scanning electrochemical microscopy

This paper describes a new approach for quantification of rates of molecular transport through patterned, or otherwise heterogeneous, porous films supported on conductive substrates. Scanning Electrochemical Microscopy (SECM) has been used to image molecular sieving of redox active probes by thin, electropolymerized films of Fe(5-amino-1,10-phenanthroline)3(2+) on micropatterned and microdisk array electrodes. Films as thin as 12 nm completely block redox mediators with average molecular diameters greater than 12 A, whereas smaller diameter probes (radii 5-8 A) were observed to permeate selectively. SECM tip currents measured for three different redox permeants/mediators are observed to decrease with increasing polymer thickness, consistent with a transport model that includes partitioning into and diffusion within the polymer films. Permeabilities, PDf, within the poly[Fe(5-NH2-phen)3(2+)] films have been quantitatively determined from the SECM tip currents and are in excellent agreement with data previously obtained from rotatingdisk electrochemistry. This new methodology provides a versatile approach for quantitative investigation of membrane transport and permeation selectivity with good lateral spatial resolution.     

Mixed-mode anion-cation exchange/hydrophilic interaction liquid chromatography-electrospray mass spectrometry as an alternative to reversed phase for small molecule drug discovery

Within pharmaceutical drug discovery, significant needs currently exist for the analysis and purification of structurally diverse samples prior to or immediately following high-throughput screening. These processes are required to facilitate rapid and accurate biological profiling, structural determination, and resupply of new drug candidates. Reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) for both analytical and preparative applications has become the small molecule separation/detection tool of choice for meeting many of these needs. However, the separation selectivity provided by RP-HPLC has been limited to the hydrophobicity-based resolution of relatively nonpolar sample components, and for high-throughput drug discovery applications, no sufficient alternative procedures have been identified. In this investigation, a mixed-mode anion-cation exchange/hydrophilic interaction chromatography (ACE-HILIC) method has been developed to provide both direct compatibility with ESI-MS and evaporative light-scattering detection (ELSD) and separation selectivity highly orthogonal to RP-HPLC. The technique employed silica-based small-pore weak ion exchange resins eluted with a combined aqueous and pH gradient. A diverse set of dipeptide probes was employed for the elucidation of the relative contributions of three retention mechanisms. ACE-HILIC-ESI-MS-ELSD should prove useful for the analysis and purification of compounds from both biological (e.g., natural products) and synthetic (e.g., combinatorial chemistry) sources of molecular diversity.     

Sintering mechanisms in strontium doped lanthanum chromite

The sintering behavior of (La_0.7Sr_0.3)_xCrO₃ (0.95 x 1.05) is investigated to compare liquid phase sintering phenomena occuring in stoichiometric and non-stoichiometric compositions. Shrinkage analysis revealed marked contrast between the densification characteristics of the A-site enriched (x > 1.00) and A-site depleted (x < 1.00) materials. A-site depleted samples typically exhibited a single liquid phase sintering event at 1250 °C attributed to the melting of an exsoluted SrCrO₄ phase. A-site enriched samples indicated two rapid shrinkage events due to the melting of SrCrO₄, and a Sr_2.67(CrO₄)₂ phase with a melting temperature of 1450 °C. Sr_2.67(CrO₄)₂ was shown to evolve from a decomposition reaction between SrCrO₄ and La₂CrO₆, detected together in A-site enriched samples from 800–1000 °C. Maximum densities (93% theoretical density) were achieved for (La_0.7Sr_0.3)_xCrO₃ x = 1.00 after sintering at 1700 °C for two hours.     

Proteolytic cleavage and activation of PAK2 during UV irradiation-induced apoptosis in A431 cells

Exposure of mammalian cells to ultraviolet (UV) light elicits a cellular response and can also lead to apoptotic cell death. In this report, we show that a 36-kDa myelin basic protein (MBP) kinase detected by an in-gel kinase assay can be dramatically activated during the early stages of UV irradiation-triggered apoptosis of A431 cells. Immunoblot analysis revealed that this 36-kDa MBP kinase could be recognized by an antibody against the C-terminal regions of a family of p21Cdc42/Rac-activated kinases (PAKs). By using this antibody and a PAK2-specific antibody against the N-terminal region of PAK2 as studying tools, we further demonstrated that UV irradiation caused cleavage of PAK2 to generate a 36-kDa C-terminal catalytic fragment and a 30-kDa N-terminal fragment in A431 cells. The appearance of the 36-kDa C-terminal catalytic fragment of PAK2 matched exactly with the activation of the 36-kDa MBP kinase in A431 cells upon UV irradiation. In addition, UV irradiation also led to activation of CPP32/caspase-3, but not ICH-1L/caspase-2 and ICE/caspase-1, in A431 cells and the kinetics of activation of CPP32/caspase-3 appeared to correlate well with that of DNA fragmentation and of cleavage/activation of PAK2, respectively. Moreover, blockage of activation of CPP32/caspase-3 by pretreating the cells with two specific tetrapeptidic inhibitors for caspases (Ac-DEVD-cho and Ac-YVAD-cmk) could significantly attenuate the extent of cleavage/activation of PAK2 induced by UV irradiation. Collectively, the results demonstrate that cleavage and activation of PAK2 can be induced during the early stages of UV irradiation-triggered apoptosis and indicate the involvement of CPP32/caspase-3 in this process.     

Oxidative damage and fumonisin B1-induced toxicity in primary rat hepatocytes and rat liver in vivo

The particle size distribution and the metal speciation of the heavy metals were investigated on dredged sediment and on the fractions obtained by mechanical agitated (Denver) flotation. The transition metal ions (cadmium, copper, lead and zinc) were flotated specifically independent of the particle size. Particle size analysis, EDTA extraction and sequential extracts indicated that during flotation a redistribution of metals occurred due to the oxidation of metal sulphides. This oxidation process was more pronounced when the flotation was performed at higher pH values and resulted in a decrease in flotation specificity.     

Screening and chemoprophylaxis for tuberculosis infection in college populations

Lyme borreliosis is the most frequent tick-borne disease in the Northern hemisphere. Here we describe the first isolation of Borrelia burgdorferi sensu lato in Bulgaria: the midguts of 47 Ixodes ricinus obtained by flagging from the Central park in Sofia, Bulgaria were cultivated for borreliae in BSK medium. The eight isolates obtained from the ticks and one skin isolate from a Bulgarian patient with erythema migrans were subjected to phenotypic [outer surface protein A (OspA) serotyping] and genotypic analysis (pulsed-field gel electrophoresis typing followed by large restriction fragment pattern analysis after MluI digestion, polymerase chain reaction with 16S rRNA-directed oligonucleotide probes, and restriction fragment length polymorphism analysis of rrf-rrl intergenic spacer amplicons). The skin isolate was B. burgdorferi sensu stricto, as were four of the tick isolates; the other four tick isolates were B. garinii representing three different OspA serotypes (types 3, 5 and 7). These findings confirm the wide geographic distribution of the different B. garinii-associated OspA serotypes in Europe (shown here for the first time for the Southeastern part of Europe) and of B. burgdorferi sensu stricto in the Western hemisphere. These findings have implications for development of diagnostic tests and a borrelia vaccine in Southeastern Europe.     

Low-dose oral etoposide monotherapy in adult Langerhans cell histiocytosis

BACKGROUND: The purpose of this study was to test the disease-controlling effect of low-dose oral etoposide monotherapy in adult-onset multisystem Langerhans cell histiocytosis. There are no previous reports of low-dose etoposide monotherapy for this condition. OBSERVATIONS: A 27-year-old man with a 7-year history of multifocal chronic Langerhans cell histiocytosis presented with severe disabling ulcers in intertriginous areas. He had previously been treated with 2 different regimens of antitumoral chemotherapy; one had to be discontinued due to myelosuppression and the other had proved ineffective. We treated with oral etoposide monotherapy at 50 mg/d (22 mg/m2 per day) for 21 days. The treatment was repeated at 28-day intervals for a total of 6 cycles. A rapid initial response with subtotal diminution of the involved skin area was found. No adverse effects were observed. The clinical picture has remained stable during the 7 months following cessation of therapy. CONCLUSION: Low-dose oral etoposide treatment is an adequate therapeutic measure for prolonged disease control in adult-type Langerhans cell histiocytosis.     

Effect of ischaemia and reperfusion on the intracellular concentration of taurine and glutamine in the hearts of patients undergoing coronary artery surgery

Taurine and glutamine are the most abundant intracellular free amino acids in mammalian hearts where changes in their intracellular concentrations are likely to influence a number of cellular activities. In this study we investigated the effects of ischaemia and reperfusion on the intracellular concentrations of taurine and glutamine in the hearts of patients undergoing coronary artery bypass surgery using cold crystalloid or cold blood cardioplegic solutions. Ischaemic arrest (30 min), using cold crystalloid cardioplegic solution (n = 19), decreased the intracellular concentrations (micromol/g wet weight) of taurine (from 9.8 +/- 0.8 to 7.7 +/- 0.7, P < 0.05) and glutamine (8.7 +/- 0.5 to 7.2 +/- 0.6). After 20 min of normothermic reperfusion the fall in taurine and glutamine was maintained (7.5 +/- 0.5 and 7.4 +/- 0.7 for taurine and glutamine respectively). Myocardial ischaemic arrest with cold blood cardioplegic solution (n = 16) did not cause a significant fall in tissue taurine or glutamine. However, on reperfusion there was a marked fall in the intracellular concentrations of taurine (9.4 +/- 0.5 to 6.5 +/- 0.7) and glutamine (8.0 +/- 0.7 to 5.8 +/- 0.4). The fall in amino acids was associated with a fall in ATP and a rise in tissue lactate. This work demonstrates that irrespective of the cardioplegic solution used to arrest the heart, there is a marked fall in tissue taurine and glutamine which may influence the extent of recovery following surgery. The fall in taurine is largely due to efflux whereas changes in glutamine are due to both transport and metabolism. Ischaemia, hypothermia and changes in the transmembrane concentration gradients are the likely factors responsible for the changes in tissue amino acids.     

Characterization of the native and recombinant catalytic subunit of human DNA polymerase gamma: identification of residues critical for exonuclease activity and dideoxynucleotide sensitivity

The human DNA polymerase gamma catalytic subunit was overexpressed in recombinant baculovirus-infected insect cells, and the 136 000 Da protein was purified to homogeneity. Application of the same purification protocol to HeLa mitochondrial lysates permitted isolation of native DNA polymerase gamma as a single subunit, allowing direct comparison of the native and recombinant enzymes without interference of other polypeptides. Both forms exhibited identical properties, and the DNA polymerase and 3' --> 5' exonuclease activities were shown unambiguously to reside in the catalytic polypeptide. The salt sensitivity and moderate processivity of the isolated catalytic subunit suggest other factors could be required to restore the salt tolerance and highly processive DNA synthesis typical of gamma polymerases. To facilitate our understanding of mitochondrial DNA replication and mutagenesis as well as cytotoxicity mediated by antiviral nucleotide analogues, we also constructed two site-directed mutant proteins of the human DNA polymerase gamma. Substituting alanine for two essential acidic residues in the exonuclease motif selectively eliminated the 3' --> 5' exonucleolytic function of the purified mutant polymerase gamma. Replacement of a tyrosine residue critical for sugar recognition with phenylalanine in polymerase motif B reduced dideoxynucleotide inhibition by a factor of 5000 with only minor effects on overall polymerase function.