Transforming Rhodamine Dyes for (d)STORM Super-Resolution Microscopy via 1,3-Disubstituted Imidazolium Substitution.

We introduce a strategy to optimize the photoswitching behavior of rhodamines for (d)STORM super-resolution microscopy. By replacing the benzene ring in the rhodamine core with a permanently charged 1,3-disubstituted imidazolium, the resultant dyes are markedly sensitized toward photoswitching, and exhibit outstanding (d)STORM performance with fast on-off switching, long-lasting blinking, and bright single-molecule emission. We thus attain excellent (d)STORM images under green excitation that are on par with the "ideal" red-excited dyes, including for difficult structures such as the mammalian actin cytoskeleton, and demonstrate high-quality two-color three-dimensional (d)STORM.

Identification of adenine nucleotide translocase 4 inhibitors by molecular docking.

The protein adenine nucleotide translocase (ANT) is localized in the mitochondrial inner membrane and plays an essential role in transporting ADP into the mitochondrial matrix and ATP out from the matrix for cell utilization. In mammals there are four paralogous ANT genes, of which ANT4 is exclusively expressed in meiotic germ cells. Since ANT4 has been shown essential for spermatogenesis and male fertility in mice, inhibition of ANT4 appears to be a reasonable target for male contraceptive development. Further, in contrast to ANT1, ANT2 and ANT3 that are highly homologous to each other, ANT4 has a distinguishable amino acid sequence, which serves as a basis to develop a selective ANT4 inhibitor. In this study, we aimed to identify candidate compounds that can selectively inhibit ANT4 activity over the other ANTs. We used a structure-based method in which ANT4 was modeled then utilized as the basis for selection of compounds that interact with sites unique to ANT4. A large chemical library (>100,000 small molecules) was screened by molecular docking and effects of these compounds on ADP/ATP exchange through ANT4 were examined using yeast mitochondria expressing human ANT4. Through this, we identified one particular candidate compound, [2,2'-methanediylbis(4-nitrophenol)], which inhibits ANT4 activity with a lower IC50 than the other ANTs (5.8 µM, 4.1 µM, 5.1 µM and 1.4 µM for ANT1, 2, 3 and 4, respectively). This newly identified active lead compound and its chemical structure are expected to provide new opportunities to optimize selective ANT4 inhibitors for contraceptive purposes.

Functional expression of human adenine nucleotide translocase 4 in Saccharomyces cerevisiae.

The adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP across the inner mitochondrial membrane. The human genome encodes multiple ANT isoforms that are expressed in a tissue-specific manner. Recently a novel germ cell-specific member of the ANT family, ANT4 (SLC25A31) was identified. Although it is known that targeted depletion of ANT4 in mice resulted in male infertility, the functional biochemical differences between ANT4 and other somatic ANT isoforms remain undetermined. To gain insight into ANT4, we expressed human ANT4 (hANT4) in yeast mitochondria. Unlike the somatic ANT proteins, expression of hANT4 failed to complement an AAC-deficient yeast strain for growth on media requiring mitochondrial respiration. Moreover, overexpression of hANT4 from a multi-copy plasmid interfered with optimal yeast growth. However, mutation of specific amino acids of hANT4 improved yeast mitochondrial expression and supported growth of the AAC-deficient yeast on non-fermentable carbon sources. The mutations affected amino acids predicted to interact with phospholipids, suggesting the importance of lipid interactions for function of this protein. Each mutant hANT4 and the somatic hANTs exhibited similar ADP/ATP exchange kinetics. These data define common and distinct biochemical characteristics of ANT4 in comparison to ANT1, 2 and 3 providing a basis for study of its unique adaptation to germ cells.

OS-FRET: a new one-sample method for improved FRET measurements.

Fluorescence resonance energy transfer (FRET) is a powerful tool for studying macromolecular assemblies in vitro under near-physiological conditions. Here we present a new type of one-sample FRET (OS-FRET) method employing a novel, nonfluorescent methanethiosulfonate-linked acceptor that can be reversibly coupled to a target sulfhydryl residue via a disulfide bond. After the quenched donor emission is quantitated, the acceptor is removed by reduction, allowing measurement of unquenched donor emission in the same sample. Previous one-sample methods provide distinct advantages in specific FRET applications. The new OS-FRET method is a generalizable spectrochemical approach that can be applied to macromolecular systems lacking essential disulfide bonds and eliminates the potential systematic errors of some earlier one-sample methods. In addition, OS-FRET enables quantitative FRET measurements in virtually any fluorescence spectrometer or detection device. Compared to conventional multisample FRET methods, OS-FRET conserves sample, increases the precision of data, and shortens the time per measurement. The utility of the method is illustrated by its application to a protein complex of known structure formed by CheW and the P4-P5 fragment of CheA, both from Thermotoga maritima. The findings confirm the practicality and advantages of OS-FRET. Anticipated applications of OS-FRET include analysis of macromolecular structure, binding and conformational dynamics, and high-throughput screening for interactions and inhibitors.

Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications.

BACKGROUND: EvaGreen (EG) is a newly developed DNA-binding dye that has recently been used in quantitative real-time PCR (qPCR), post-PCR DNA melt curve analysis and several other applications. However, very little is known about the physicochemical properties of the dye and their relevance to the applications, particularly to qPCR and post PCR DNA melt curve analysis. In this paper, we characterized EG along with a widely used qPCR dye, SYBR Green I (SG), for their DNA-binding properties and stability, and compared their performance in qPCR under a variety of conditions. RESULTS: This study systematically compared the DNA binding profiles of the two dyes under different conditions and had these findings: a) EG had a lower binding affinity for both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) than SG; b) EG showed no apparent preference for either GC- or AT-rich sequence while SG had a slight preference for AT-rich sequence; c) both dyes showed substantially lower affinity toward ssDNA than toward dsDNA and even lower affinity toward shorter ssDNA fragments except that this trend was more pronounced for EG. Our results also demonstrated that EG was stable both under PCR condition and during routine storage and handling. In the comparative qPCR study, both EG and SG exhibited PCR interference when used at high dye concentration, as evident from delayed Ct and/or nonspecific product formation. The problem worsened when the chain extension time was shortened or when the amplicon size was relatively long (>500 bp). However, qPCR using EG tolerated a significantly higher dye concentration, thus permitting a more robust PCR signal as well as a sharper and stronger DNA melt peak. These differences in qPCR performance between the two dyes are believed to be attributable to their differences in DNA binding profiles. CONCLUSION: These findings suggest that an ideal qPCR dye should possess several DNA-binding characteristics, including a "just right" affinity for dsDNA and low or no affinity for ssDNA and short DNA fragments. The favorable DNA-binding profile of EG, coupled with its good stability and instrument-compatibility, should make EG a promising dye for qPCR and related applications.

Argon laser peripheral iridoplasty in the management of phacomorphic glaucoma.

BACKGROUND AND OBJECTIVE: To document the efficacy and safety of argon laser peripheral iridoplasty (ALPI) as a temporizing measure in the treatment of phacomorphic glaucoma and to evaluate the mid-term visual outcomes. PATIENTS AND METHODS: The clinical records of all patients with phacomorphic glaucoma treated with ALPI from December 1999 to December 2002 were retrospectively reviewed. The primary outcome measure was the rate of successful control of intraocular pressure. Successful cases with at least 12 months of follow-up were further analyzed regarding their outcomes after cataract surgery, including visual acuity, intraocular pressure (IOP), and angle status. RESULTS: ALPI was performed as an initial step in the management of phacomorphic glaucoma in 21 eyes of 21 patients. IOP was successfully controlled in 17 eyes (80.75%), with a statistically significant decrease at 2 and 24 hours postoperatively (P < .0001). Elective cataract surgery was performed in 16 of the 17 successful cases and 12 of these patients completed at least 12 months of follow-up. After a mean follow-up period of 25.58 months, IOP was normal without medications in 11 eyes and only 1 patient required antiglaucoma eye drops to control IOP. All patients achieved a visual acuity of at least 1/60 and 8 (66.67%) of them achieved a visual acuity of 6/21 or better. All patients maintained an open angle without further surgical intervention. No complication was noted directly attributable to ALPI. CONCLUSION: ALPI is a safe and efficacious measure for the initial management of phacomorphic glaucoma, simultaneously obviating the need to operate in highly inflamed eyes in an emergency setting and achieving satisfactory mid-term visual outcome.

Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology.

Systematic parallel analysis of the phosphorylation status of networks of interacting proteins involved in the regulatory circuitry of cells and tissues is certain to drive research in the post-genomics era for many years to come. Reversible protein phosphorylation plays a critical regulatory role in a multitude of cellular processes, including alterations in signal transduction pathways related to oncogene and tumor suppressor gene products in cancer. While fluorescence detection methods are likely to offer the best solution to global protein quantitation in proteomics, to date, there has been no satisfactory method for the specific and reversible fluorescent detection of gel-separated phosphoproteins from complex samples. The newly developed Pro-Q Diamond phosphoprotein dye technology is suitable for the fluorescent detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl sulfate (SDS)-polyacrylamide gels and two-dimensional (2-D) gels. Additionally, the technology is appropriate for the determination of protein kinase and phosphatase substrate preference. Other macromolecules, such as DNA, RNA, and sulfated glycans, fail to be detected with Pro-Q Diamond dye. The staining procedure is rapid, simple to perform, readily reversible and fully compatible with modern microchemical analysis procedures, such as matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Pro-Q Diamond dye technology can detect as little as 1-2 ng of beta-casein, a pentaphosphorylated protein, and 8 ng of pepsin, a monophosphorylated protein. Fluorescence signal intensity correlates with the number of phosphorylated residues on the protein. Through combination of Pro-Q Diamond phosphoprotein stain with SYPRO(R) Ruby protein gel stain, Multiplexed Proteomics technology permits quantitative, dichromatic fluorescence detection of proteins in 2-D gels. This evolving discovery platform allows the parallel determination of protein expression level changes and altered post-translational modification patterns within a single 2-D gel experiment. The linear responses of the fluorescence dyes utilized, allow rigorous quantitation of changes over an unprecedented 500-1000-fold concentration range.

Simultaneous trichromatic fluorescence detection of proteins on Western blots using an amine-reactive dye in combination with alkaline phosphatase- and horseradish peroxidase-antibody conjugates.

Three-color fluorescence detection methods are described based upon covalently coupling the dye 2-methoxy-2,4-diphenyl-2(2H)-furanone (MDPF) to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using alkaline-phosphatase-conjugated reporter molecules in combination with the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO-phosphate) as well as horseradish peroxidase-conjugated reporter molecules in combination with the new fluorogenic substrate Amplex Gold reagent. This results in all proteins in the profile being visualized as fluorescent blue signal, those detected specifically with the alkaline phosphatase conjugate appearing as fluorescent red signal and those detected specifically with the horseradish peroxidase conjugate appearing as fluorescent yellow signal. Using conventional secondary antibodies, two different targets may be identified as long as primary antibodies generated from two different species are used in the analysis. However, Zenon antibody labeling technology eliminates this restriction, permitting the simultaneous use of two different mouse monoclonal antibodies or two different rabbit polyclonal antibodies in the same electroblotting experiment. The trichromatic detection system is broadly compatible with UV epi-illuminators combined with photographic or charge-coupled device (CCD) cameras, and xenon-arc sources equipped with appropriate excitation/emission filters. Alternatively, the enzyme conjugates may be detected using a laser-based gel scanner. The trichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of two different target proteins relative to the entire protein profile on a single electroblot, precluding any requirement for running replicate gels that would otherwise require separate visualization of total proteins and subsequent alignment with multiple chemiluminescent or colorimetric signals generated on different electroblots.

Detection of glycoproteins in polyacrylamide gels and on electroblots using Pro-Q Emerald 488 dye, a fluorescent periodate Schiff-base stain.

Pro-Q Emerald 488 glycoprotein stain reacts with periodic acid-oxidized carbohydrate groups, generating a bright green-fluorescent signal on glycoproteins. The stain permits detection of less than 5-18 ng of glycoprotein per band, depending upon the nature and the degree of protein glycosylation, making it roughly 8-16-fold more sensitive than the standard colorimetric periodic acid-Schiff base method using acidic fuchsin dye (pararosaniline). The green-fluorescent signal from Pro-Q Emerald 488 stain may optimally be visualized using charge-coupled device/xenon arc lamp-based imaging systems or 470-488 nm laser-based gel scanners. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and the specificity of staining is better in gels. After detecting glycoproteins with Pro-Q Emerald 488 dye, total protein profiles may subsequently be detected using SYPRO Ruby protein gel stain. Using computer-assisted registration techniques, images may then be merged to generate differential display maps.

Fluorescence detection and quantitation of recombinant proteins containing oligohistidine tag sequences directly in sodium dodecyl sulfate-polyacrylamide gels.

Two fluorophore-nitrilotriacetic acid conjugates, Pro-Q Sapphire 365 and Pro-Q Sapphire 488 oligohistidine gel stains, have been developed for the fluorescence detection of fusion proteins containing oligohistidine tags directly in sodium dodecyl sulfate polyacrylamide gels, without the requirement for electroblotting, reporter enzymes or secondary detection reagents. Pro-Q Sapphire 365 oligohistidine gel stain exhibits bright-blue fluorescence (emission maximum = 450 nm) when illuminated with UV-A or UV-B light from a standard ultraviolet transilluminator. Pro-Q Sapphire 488 oligohistidine gel stain exhibits bright-green fluorescence (emission maximum = 515 nm) when illuminated with visible light from a laser-based gel scanner equipped with a 470 nm second-harmonic generation (SHG) or 488 nm argon-ion laser source. Typically, 25-65 ng of oligohistidine-tagged fusion protein in whole cell lysates is detectable using either stain. After documenting the fluorescence signal from the Pro-Q Sapphire dyes, gels may be post-stained with the red-fluorescent SYPRO Ruby protein gel stain in order to reveal the total protein pattern.