Excitation and emission wavelength ratiometric cyanide-sensitive probes for physiological sensing

We characterize three new fluorescent probes that show both spectral shifts and intensity changes in the presence of aqueous cyanide, allowing for both excitation and fluorescence emission wavelength ratiometric and colorimetric sensing. The relatively high binding constants of the probes for cyanide enables a distinct colorimetric change to be visually observed with as little as 10 microM cyanide. The response of the new probes is based on the ability of the boronic acid group to interact with the CN(-) anion, changing from the neutral form of the boronic acid group R-B(OH)(2) to the anionic R-B(-)(OH)3 form, which is an electron-donating group. The presence of an electron-deficient quaternary heterocyclic nitrogen center and a strong electron-donating amino group in the 6 position on the quinolinium backbone provides for the spectral changes observed upon CN(-) complexation. We have determined the binding constants for the ortho-, meta-, and para-boronic acid probes to be 0.12, 0.17, and 0.14 microM(-3). In addition we have synthesized a control compound that does not contain the boronic acid moiety, allowing for structural comparisons and a rationale for the sensing mechanism to be made. Finally we show that the affinity for monosaccharides, such as glucose or fructose, is relatively low as compared to that for cyanide, enabling the potential detection of cyanide in physiologies up to lethal levels.     

Boronic acid fluorescent sensors for monosaccharide signaling based on the 6-methoxyquinolinium heterocyclic nucleus: progress toward noninvasive and continuous glucose monitoring

The synthesis, characterization, and spectral properties of strategically designed boronic acid containing fluorescent sensors, o-, m-, p-BMOQBA, for the potential detection of tear glucose concentrations when immobilized in plastic disposable contact lenses is described. The new probes, BMOQBAs, consist of the 6-methoxyquinolinium nucleus as a fluorescent indicator, and the boronic acid moiety as a glucose chelating group. A control compound BMOQ, which has no boronic acid group and therefore does not bind monosaccharides has also been prepared. In this paper, we show that structural design considerations of the new probes have afforded for their compatibility within the lenses, with reduced probe sugar-bound pK(a) favorable with the mildly acidic lens environment. In addition, the new probes are readily water soluble, have high quantum yields, and can be prepared by a simple one-step synthetic procedure.     

Formation of 8-methylguanine as a result of DNA alkylation by methyl radicals generated during horseradish peroxidase-catalyzed oxidation of methylhydrazine

Methylhydrazine oxidation promoted by horseradish peroxidase-H2O2 or ferricyanide led to the generation of high yields of methyl radicals and to the formation of 7-methylguanine and 8-methylguanine upon interaction with calf thymus DNA. Methyl radicals were identified by spin-trapping experiments with alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone and tert-nitrosobutane. The methylated guanine products were identified in the neutral hydrolysates of treated DNA by high pressure liquid chromatography (HPLC) analysis and spiking with authentic samples. The structure of 8-methylguanine, a product not previously reported in enzymatic systems, was confirmed by HPLC chromatography, UV absorbance, and mass spectrometry. The formation of 8-methylguanine suggests a possible role for carbon-centered radicals as DNA-alkylating agents.     

N terminus of calpain 1 is a mitochondrial targeting sequence

The ubiquitous m- and mu-calpains are thought to be localized in the cytosolic compartment, as is their endogenous inhibitor calpastatin. Previously, mu-calpain was found to be enriched in mitochondrial fractions isolated from rat cerebral cortex and SH-SY5Y neuroblastoma cells, but the submitochondrial localization of mu-calpain was not determined. In the present study, submitochondrial fractionation and digitonin permeabilization studies indicated that both calpain 1 and calpain small subunit 1, which together form mu-calpain, are present in the mitochondrial intermembrane space. The N terminus of calpain 1 contains an amphipathic alpha-helical domain, and is distinct from the N terminus of calpain 2. Calpain 1, but not calpain 2, was imported into mitochondria. Removal of the N-terminal 22 amino acids of calpain 1 blocked the mitochondrial calpain import, while addition of this N-terminal region to calpain 2 or green fluorescent protein enabled mitochondrial import. The N terminus of calpain 1 was not processed following mitochondrial import, but was removed by autolysis following calpain activation. Calpain small subunit 1 was not directly imported into mitochondria, but was imported in the presence of calpain 1. The presence of a mitochondrial targeting sequence in the N-terminal region of calpain 1 is consistent with the localization of mu-calpain to the mitochondrial intermembrane space and provides new insight into the possible functions of this cysteine protease.     

Drosophila Heterochromatin Protein 1 (HP1)/Origin Recognition Complex (ORC) Protein Is Associated with HP1 and ORC and Functions in Heterochromatin-induced Silencing

Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.     

A glucose-sensing contact lens: from bench top to patient

In the past few years we have seen the development of several new technologies for the continuous and non-invasive monitoring of physiological glucose, such as the GlucoWatch, glucose-sensing skin patches and approaches based on a glucose-sensing tattoo. One approach that differs from current thinking is based on the determination and monitoring of tear glucose, which is well known to track blood glucose with an approximate 30 min lag time, using disposable and colorless contact lenses. These contact lenses can be worn by diabetics who can colorimetrically see changes in their contact lens color or other fluorescence-based properties, giving an indication of tear and blood glucose levels.     

Identification of C8-methylguanine in the hydrolysates of DNA from rats administered 1,2-dimethylhydrazine. Evidence for in vivo DNA alkylation by methyl radicals.

C8-Methylguanine was identified in the neutral hydrolysates of DNA isolated from the liver or colon tissue of rats administered 1,2-dimethylhydrazine. In all the samples examined, the biologically isolated adducts were characterized by co-elution with synthetic C8-methylguanine under different high pressure liquid chromatography conditions. The sample isolated from liver DNA was also identified by UV spectroscopy at different pH values and by mass spectrometry. The estimated yields of C8-methylguanine obtained in hydrolysates of DNA from the liver or colon tissue were comparable to those of O6-methylguanine. C8-Methylguanine was not detected when the spin trap alpha-(4-pyridyl-1-oxide)-N-tert- butylnitrone was administered together with 1,2-dimethylhydrazine. The spin trap also inhibited N7-methylguanine and O6-methylguanine yields, although to a lesser extent. These results constitute the first evidence that DNA alkylation by carbon-centered radicals can occur in vivo.     

Fluorescence Quenching of CdTe Nanocrystals by Bound Gold Nanoparticles in Aqueous Solution

Water-soluble gold nanoparticles with an average diameter of 5 nm were prepared with carboxylic acid terminated thiol ligands. These ligands contain zero to eight methylene moieties. CdTe nanocrystals with an average diameter of 5 nm were synthesized with aminoethanethiol capping. These nanocrystals displayed characteristic absorption and emission spectra of quantum dots. The amine terminated CdTe nanocrystals and carboxylic-acid-terminated gold nanoparticles were conjugated in aqueous solution at pH 5.0 by electrostatic interaction, and the conjugation was monitored with fluorescence spectroscopy. The CdTe nanocrystals were significantly quenched upon binding with gold nanoparticles. The quenching efficiency was affected by both the concentration of gold nanoparticles in the complex and the length of spacer between the CdTe nanocrystal and Au nanoparticle. The observed quenching was explained using Förster resonance energy transfer (FRET) mechanism, and the Förster distance was estimated to be 3.8 nm between the donor–acceptor pair.     

Radiative decay engineering 6: Fluorescence on one-dimensional photonic crystals

During the past decade the interactions of fluorophores with metallic particles and surfaces has become an active area of research. These near-field interactions of fluorophores with surface plasmons have resulted in increased brightness and directional emission. However, using metals has some disadvantages such as quenching at short fluorophore–metal distances and increased rates of energy dissipation due to lossy metals. These unfavorable effects are not expected in dielectrics. In this article, we describe the interactions of fluorophores with one-dimensional (1D) photonic crystals (PCs), which have alternating layers of dielectrics with dimensions that create a photonic band gap (PBG). Freely propagating light at the PBG wavelength will be reflected. However, similar to metals, we show that fluorophores within near-field distances of the 1DPC interacts with the structure. Our results demonstrate that these fluorophores can interact with both internal modes and Bloch surface waves (BSWs) of the 1DPC. For fluorophores on the surface of the 1DPC, the emission dominantly occurs through the 1DPC and into the substrate. We refer to these two phenomena together as Bragg grating-coupled emission (BGCE). Here we describe our preliminary results on BGCE. 1DPCs are simple to fabricate and can be handled and reused without damage. We believe that BGCE provides opportunities for new formats for fluorescence detection and sensing.     

Identification of Plasmodium falciparum DNA Repair Protein Mre11 with an Evolutionarily Conserved Nuclease Function

The eukaryotic Meiotic Recombination protein 11 (Mre11) plays pivotal roles in the DNA damage response (DDR). Specifically, Mre11 senses and signals DNA double strand breaks (DSB) and facilitates their repair through effector proteins belonging to either homologous recombination (HR) or non-homologous end joining (NHEJ) repair mechanisms. In the human malaria parasite Plasmodium falciparum, HR and alternative-NHEJ have been identified; however, little is known about the upstream factors involved in the DDR of this organism. In this report, we identify a putative ortholog of Mre11 in P. falciparum (PfalMre11) that shares 22% sequence similarity to human Mre11. Homology modeling reveals striking structural resemblance of the predicted PfalMre11 nuclease domain to the nuclease domain of Saccharomyces cerevisiae Mre11 (ScMre11). Complementation analyses reveal functional conservation of PfalMre11 nuclease activity as demonstrated by the ability of the PfalMre11 nuclease domain, in conjunction with the C-terminal domain of ScMre11, to functionally complement an mre11 deficient yeast strain. Functional complementation was virtually abrogated by an amino acid substitution in the PfalMre11 nuclease domain (D398N). PfalMre11 is abundant in the mitotically active trophozoite and schizont stages of P. falciparum and is up-regulated in response to DNA damage, suggesting a role in the DDR. PfalMre11 exhibits physical interaction with PfalRad50. In addition, yeast 2-hybrid studies show that PfalMre11 interacts with ScRad50 and ScXrs2, two important components of the well characterized Mre11-Rad50-Xrs2 complex which is involved in DDR signaling and repair in S. cerevisiae, further supporting a role for PfalMre11 in the DDR. Taken together, these findings provide evidence that PfalMre11 is an evolutionarily conserved component of the DDR in Plasmodium.