Development of High-Throughput Method for Measurement of Vascular Nitric Oxide Generation in Microplate Reader

Background: Despite the importance of nitric oxide (NO) in vascular physiology and pathology, a high-throughput method for the quantification of its vascular generation is lacking. Objective: By using the fluorescent probe 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM), we have optimized a simple method for the determination of the generation of endothelial nitric oxide in a microplate format. Methods: A nitric oxide donor was used (3-morpholinosydnonimine hydrochloride, SIN-1). Different factors affecting the method were studied, such as the effects of dye concentration, different buffers, time of reaction, gain, and number of flashes. Results: Beer’s law was linear over a nanomolar range (1–10 nM) of SIN-1 with wavelengths of maximum excitation and emission at 495 and 525 nm; the limit of detection reached 0.897 nM. Under the optimized conditions, the generation of rat aortic endothelial NO was measured by incubating DAF-FM with serial concentrations (10–1000 µM) of acetylcholine (ACh) for 3 min. To confirm specificity, Nω-Nitro-l-arginine methyl ester (l-NAME)—the standard inhibitor of endothelial NO synthase—was found to inhibit the ACh-stimulated generation of NO. In addition, vessels pre-exposed for 1 h to 400 µM of the endothelial damaging agent methyl glyoxal showed inhibited NO generation when compared to the control stimulated by ACh. Conclusions: The capability of the method to measure micro-volume samples makes it convenient for the simultaneous handling of a very large number of samples. Additionally, it allows samples to be run simultaneously with their replicates to ensure identical experimental conditions, thus minimizing the effect of biological variability

Polymer-Enhanced Capillary Transient Isotachophoresis with Boronic Acid-Functionalized Squarylium Dyes for the Fluorescent Determination of Digoxin and Digoxigenin

<p>Here is reported a new application of polymer-enhanced capillary transient isotachophoresis for the separation and quantification of the drug digoxin and its primary metabolite digoxigenin coupled with laser-induced fluorescence (LIF) detection facilitated by labeling with two boronic acid-functionalized squarylium dyes of different alkyl side chain lengths, SQ-BA1 and SQ-BA2. The conditions for drug–dye complex formation were optimized, as determined by absorbance and fluorescence spectra, according to solution pH and buffer composition. As digoxin has a digitoxose sugar moiety in its structure, it was shown to exhibit better enhancement in the fluorescence intensity of both dyes than digoxigenin, which lacks this moiety, presumably through the formation of a cyclic boronate ester complex. A comparison of analyte labeling in pre-column and on-column modes was conducted in subsequent capillary electrophoresis-LIF studies, with the latter labeling mode yielding superior sensitivity. However, to achieve the complete resolution of labeled digoxin and digoxigenin analytes, it was necessary to use the modified isotachophoresis method, with added borate ions that may differentially interact with the drug and its metabolite, hence affecting their mobilities. Limits of quantification of the method for the determination of digoxin with SQ-BA1 and SQ-BA2 were 2.61 × 10⁻³ and 2.82 × 10⁻³ M and limits of detection were 7.83 × 10⁻⁴ and 8.47 × 10⁻⁴ M while sensitivities were as great as 5.06 × 10⁹ and 2.89 × 10⁹ M⁻¹, respectively, indicating that the method is suitable for practical analysis.</p

A Long-Wavelength Fluorescent Squarylium Cyanine Dye Possessing Boronic Acid for Sensing Monosaccharides and Glycoproteins with High Enhancement in Aqueous Solution

sensor

Carbon Dot-Mediated Capillary Electrophoresis Separations of Metallated and Demetallated Forms of Transferrin Protein

Carbon dots (CDs) are fluorescent nanomaterials used extensively in bioimaging, biosensing and biomedicine. This is due in large part to their biocompatibility, photostability, lower toxicity, and lower cost, compared to inorganic quantum dots or organic dyes. However, little is known about the utility of CDs as separation adjuvants in capillary electrophoresis (CE) separations. CDs were synthesized in-house according to a &#8216;bottom-up&#8217; method from citric acid or other simple carbon precursors. To demonstrate the applicability of CDs as separation adjuvants, mixtures of holo- (metallated) and apo- (demetallated) forms of transferrin (Tf, an iron transport protein) were analyzed. In the absence of CDs, the proteins were not resolved by a simple CE method; however, upon addition of CDs to the separation buffer, multiple forms of Tf were resolved indicating that CDs are valuable tools to facilitate the separation of analytes by CE. CE parameters including sample preparation, buffer identity, ionic strength, pH, capillary inside diameter, and temperature were optimized. The results suggest that dots synthesized from citric acid provide the best resolution of various different forms of Tf and that CDs are versatile and promising tools to improve current electrophoretic separation methods, especially for metalloprotein analysis

Manganese exposure inhibits the clearance of extracellular GABA and influenced taurine homeostasis in the striatum of developing rats.

Abstract: Manganese (Mn) accumulation in the brain has been shown to alter the neurochemistry of the basal ganglia. Mn-induced alterations in dopamine biology are fairly well understood, but recently more evidence has emerged characterizing the role of γ-aminobutyric acid (GABA) in this dysfunction. The purpose of this study was to determine if the previously observed Mninduced increase in extracellular GABA (GABA EC ) was due to altered GABA transporter (GAT) function, and whether Mn perturbs other amino acid neurotransmitters, namely taurine and glycine (known modulators of GABA). Extracellular GABA, taurine, and glycine concentrations were collected from the striatum of control (CN) or Mn-exposed Sprague-Dawley rats using in vivo microdialysis, and the GAT inhibitor nipecotic acid (NA) was used to probe GAT function. Tissue and extracellular Mn levels were significantly increased, and the Fe:Mn ratio was decreased 36-fold in the extracellular space due to Mn-exposure. NA led to a 2-fold increase in GABA EC of CNs, a response that was attenuated by Mn. Taurine responded inversely to GABA, and a novel 10-fold increase in taurine was observed after the removal of NA in CNs. Mn blunted this response and nearly abolished extracellular taurine throughout collection. Striatal taurine transporter (Slc6a6) mRNA levels were significantly increased with Mn-exposure, and Mn significantly increased 3 H-Taurine uptake after 3-min exposure in primary rat astrocytes. These data suggest that Mn increases GABA EC by inhibiting the function of GAT, and that perturbed taurine homeostasis potentially impacts neural function by jeopardizing the osmoregulatory and neuromodulatory functions of taurine in the brain. Article: INTRODUCTION An essential trace element and a cofactor for several enzymes (Hurley and Keen, 1987), manganese (Mn) is involved in immune function, regulation of metabolism, reproduction, digestion, bone growth, and blood clotting (see review by With the intriguing findings that striatal extracellular GABA (GABA EC ) concentrations are higher due to Mn-exposure , and uptake of 3 H-GABA is attenuated by Mn-exposure in striatal synaptosomes Taurine is an abundant non-essential amino acid in the brain formed from cysteine. Traditionally, brain taurine is thought to function as an osmoregulator in cells (cell volume regulation), but has also been implicated in neuromodulation, possibly functioning as a neurotransmitter. Data exist suggesting that taurine functions as an anxiolytic agent We chose to look at the taurine/GABA relationship in the striatum because it is a known region for Mn accumulation In addition to GABA and taurine, we felt it was prudent to examine the effect of Mn on another amino acid neurotransmitter, glycine. Glycine is an abundant inhibitor neurotransmitter, similar to GABA, and it is known that taurine is a glycine receptor agonist Within the brain, astrocytes are the primary cells that maintain the composition of the extracellular fluid MATERIALS AND METHODS Animals Male weanling (post-natal day 21) Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) (n = 8 for microdialysis study; n = 6 for PCR gene expression and metal analysis studies) were randomly divided into two dietary treatment groups used in previous studies Cell cultures Rat primary cortical astrocyte cultures were purchased from Invitrogen (Carlsbad, CA) and certified for purity with &gt;95% staining positive for the astrocytic marker glial fibrillary acidic protein (GFAP). Cells were grown in Dulbecco&apos;s Modified Eagle Media (D-MEM) with 15% fetal bovine serum (FBS), and maintained in a humidified atmosphere of 95% air/5% CO 2 at 37 °C. Manganese treatments were delivered using 0, 100, or 300 μM Mn in the form of MnCl 2 . These dose concentrations are based on previous studies in non-human primates reporting clinical symptoms of Mn neurotoxicity at brain concentrations of 300 μM, while 100 μM concentrations appeared to be asymptomatic H-Taurine Uptake of tritiated taurine ( 3 H-Taurine) was measured as described by Stereotaxic surgery After 5 weeks of dietary treatment and 1 week prior to microdialysis experiments, rats were anesthetized with ketamine-HCl (80 mg/kg) and xylazine (12 mg/kg) and maintained on a heating pad at 37 °C. The heads of the rats were shaved and wiped with a 5% povidone-iodine solution to reduce risk of infection. Sterile instruments and gloves were used throughout the surgical procedure. The rats were secured in the stereotaxic frame and an incision was made perpendicular to the bregma. A guide cannula (CMA/12, CMA Microdialysis, Acton, MA) was implanted into the striatum using the following coordinates: 2.4 mm lateral to the midline, 7.5 mm anterior to the lambda. The cannula was lowered to a depth of 2.5 mm, positioning it in the medial area of the striatum Microdialysis During week six of the dietary protocol, a microdialysis probe (CMA/12 Elite, CMA Microdialysis, Acton, MA) was inserted into the guide cannula and the rat was perfused with artificial cerebral spinal fluid (aCSF) (155 mM Na + , 0.83 mM Mg 2+ , 2.9 mM K + , 132.76 mM Cl − , 1.1 mM Ca + , pH 7.4) for 1 h at a flow rate of 1 μL/min. After perfusion, the flow rate was adjusted to 0.5 μL/min and 30 min fractions were collected in microtubes for a total of four and a half hours (9 samples per rat) in a refrigerated fraction collector (CMA Microdialysis, Acton, MA). This protocol has been used successfully in previous studies with stable neurotransmitter recovery in the dialysate CE-LIF analysis A protocol by RNA isolation and cDNA synthesis Total RNA was isolated from astrocyte monolayers and the striatum of control and Mn-exposed rats for quantitative PCR analysis. Tissue samples were stored in 1 mL of RNAlater ® solution (Ambion Inc., Austin, TX) and kept at −80 °C until analysis. Astrocytes were cultured in 6-well plates, then treated for 24 h with media containing 0, 100, or 300 μM Mn. Astrocytes were harvested in 500 μL denaturation solution (Ambion Inc., Austin, TX). Tissue and cell culture RNA isolation were performed using the ToTALLY RNA™ system (Ambion Inc., Austin, TX), following manufacturer&apos;s instructions. RNA concentration and purity were determined by spectrophotometric analysis before carrying out cDNA synthesis. Synthesis of cDNA was performed using the High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Foster City, CA), following manufacturer&apos;s instructions. Quantitative PCR Quantitative real-time PCR analysis was utilized to determine differential mRNA expression between control and Mn-treated tissue or cell samples of the solute carrier family taurine transporter Slc6a6 (Applied Biosystems, Foster City, CA; Rn00567962_m1, Chr. 4 -125875817-125945795). Triplicate aliquots of cDNA were analyzed on 96-well plates using TaqMan ® Gene Expression assays (Applied Biosystems, Foster City, CA). Values of cDNA expression were normalized relative to the expression of β-actin (Rn00667869_m1, Chr. 12 -12047070-12050040) analyzed from the same sample on the same plate and reported as percent of control. Metal analyses Mn, Fe, and copper (Cu) concentrations were measured with graphite furnace atomic absorption spectrometry (Varian AA240, Varian, Inc., USA). Brain tissue from the striatum was digested in ultra-pure nitric acid (1:10, w/v dilution) for 48-72 h in a sand bath (60 °C). A 50 μL aliquot of digested tissue was brought to 1 mL total volume with 2% nitric acid for analysis. The extracellular striatal samples obtained via microdialysis were not diluted due to the small volume (20 μL) and the likelihood that this biological compartment has a low concentration of metals. Bovine liver (NBS Standard Reference Material, USDC, Washington, DC) (10 μg Mn/g; 184 μg Fe/g; 80 μg Cu/g) was digested in ultra-pure nitric acid and used as an internal standard for analysis (final concentration 5 μg Mn/L; 92 μg Fe/L; 10 μg Cu/L). Statistical analysis Data were analyzed using SPSS v14 for Windows (Microsoft, Redmond, WA). Metal, baseline microdialysis, and 3 H-Taurine uptake data were analyzed using paired-samples t-tests to examine the difference between Mn-treated samples and controls. Independent sample t-tests were used to examine time-point percent change differences in the microdialysis data, time-point 3 H-Taurine uptake changes, and significance between Mn-exposed versus control mRNA expression of Scl6a6. A p-value of &lt;0.05 was considered significant. RESULTS Manganese and iron concentrations Mn-exposure resulted in significant alterations in compartmental metal concentrations. As expected, tissue Mn levels were significantly higher in Mn-exposed rats versus control (p = 0.001) ( Extracellular concentrations of taurine, GABA, and glycine Extracellular amino acid concentrations are differentially altered by Mn-exposure. Baseline levels of taurine and glycine were more abundant than GABA in the extracellular space, though Mn does not have a statistically significant effect on their levels compared to control No significant difference in baseline taurine levels was found between control and Mn-exposed animals Gly EC levels were similar in control and Mn-exposed groups, and no significant percent changes were observed between time-points within either control or Mn groups Limits of detection of the CE-LIF method employed for each neurotransmitter were found by serial dilution of derivatized standards until no discernable analyte peak could be obtained. Accordingly, limits of detection for GABA, glycine, and taurine were 6.9 ± 1.7 nM, 24 ± 5 nM, and 42 ± 21 nM, respectively, with linear dynamic ranges of 3.6 decades, 3.1 decades, and 3.3 decades, respectively. H-Taurine uptake Mn-exposure results in increased 3 H-Taurine uptake in astrocytes. After observing the unique effects of Mn-exposure on Tau EC in the striatum of rats in vivo, we decided to examine the effect of Mn-exposure on 3 H-Taurine uptake in primary rat astrocytes in vitro. Primary astrocytes exposed to Mn revealed a slight (30%) decrease in taurine uptake after 1 min, followed by a significant (219%) increase after 3 min (p = 0.034) Fig. 2: 3 H-Taurine uptake in primary astrocytes. Primary astrocytes, seeded 2 × 10 −5 in 6-well plates (n = 6) then grown to confluence, were cultured with either Mn-treated (300 μM MnCl 2 ) or control media. After 24 h cultures were exposed to 3 H-Taurine for 1, 3, or 6 min and analyzed for 3 H-Taurine retention. The inset represents percent change in uptake due to Mn-exposure expressed as percent control ± SEM. A significant (p = 0.034) increase in 3 HTaurine uptake was observed after 3 min of exposure in the Mn-treated astrocytes versus control. *p &lt; 0.05 via independent samples t-test between Mn and control treatment groups at each time-point. Gene expression of taurine transporter Mn-exposure increased taurine transporter gene expression in the rat brain, but not cultured astrocytes. Quantitative RT-PCR analysis was conducted on primary astrocytes and striatal brain tissue to determine whether or not taurine transporter (Slc6a6) gene expression reflected the observed Mn-induced alterations in Tau EC and 3 H-uptake. Chronic Mn-exposure caused a significant (p = 0.045) increase in striatal Slc6a6 mRNA levels compared to control DISCUSSION The purpose of this study was to examine the effect of Mn on GAT-mediated GABA uptake. Knowing that glycine and taurine are important amino acid neurotransmitters that are known to modulate GABA neurochemistry Fig. 4: Working model for Mn-induced GABA and taurine alterations. The dynamic shifts in neurotransmitter concentrations observed in response to nipecotic acid (NA) (panels A, B, and C) are mitigated by Mn (panels D, E, and F). We hypothesize this lack of response in Mn-exposed rats is driven by decreased GABA transporter (GAT-1) function. (A) The control panel displays GABA EC and Tau EC under normal conditions, representing baseline microdialysis measurements. All percent change (% change) comparisons in subsequent panels are based on the % change from baseline levels, represented in the control panel. Under normal conditions GABA EC binds to GABA A receptors (GABA A -R) allowing chloride ion (Cl 2− ) movement for inhibitory hyperpolarization of post-synaptic neurons, while pre-synaptic binding to GABA B receptors (GABA B -R) regulates GABA releas

On-Column Labeling of Gram-Positive Bacteria with a Boronic Acid Functionalized Squarylium Cyanine Dye for Analysis by Polymer-Enhanced Capillary Transient Isotachophoresis

A new asymmetric, squarylium cyanine dye functionalized by boronic acid (“SQ-BA”) was designed and synthesized for on-capillary labeling of gram-positive bacteria to provide for high sensitivity detection by way of a modified form of capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The CE-based separation employed a polymer-enhanced buffer with capillary transient isotachophoresis in a new hybrid method dubbed “PectI.” It was found that the addition of various monosaccharides to SQ-BA in a batch aqueous solution greatly enhanced the emission of the boronic acid functionalized dye by a factor of up to 18.3 at a long wavelength (λ_ex = 630 nm, λ_em = 660 nm) with a high affinity constant (<i>K</i> = ∼10^2.80 M^–1) superior to other sugar probes. Semiempirical quantum mechanics calculations suggest that the mechanism for this high enhancement may involve the dissociation of initially nonemissive dye associates (stabilized by an intramolecular hydrogen bond) upon complex formation with sugars. The fluorescence emission of SQ-BA was also significantly enhanced in the presence of a gram-positive bacterial spore, <i>Bacillus globigii</i> (Bg), which serves as a simulant of <i>B. anthracis</i> (or anthrax) and which possesses a peptidoglycan (sugar)-rich spore coat to provide ample sites for interaction with the dye. Several peaks were observed for a pure Bg sample even with polyethyleneoxide (PEO) present in the CE separation buffer, despite the polymer’s previously demonstrated ability to focus microoorganisms to a single peak during migration. Likewise, several peaks were observed for a Bg sample when capillary transient isotachophoresis (ctITP) alone was employed. However, the new combination of these techniques as “PectI” dramatically and reproducibly focused the bacteria to a single peak with no staining procedure. Using PectI, the trace detection of Bg spores (corresponding to approximately three cells per injection) along with separation efficiency enough to separate Bg from another gram-positive bacteria, <i>Saccharomyces cerevisiae</i> (resolution, <i>R</i>_s = 6.09, and apparent plate number, <i>N</i> = 2.7–3.3 × 10⁵), were successfully achieved