Quantification of 2-NBDG, a probe for glucose uptake, in GLUT1 overexpression in HEK293T cells by LC-MS/MS.

The growth and proliferation of most cancer cells involve the excessive uptake of glucose mediated by glucose transporters. An effective strategy for cancer therapy has been to inhibit the GLUTs that are usually overexpressed in a variety of tumor cells. 2-NBDG is a GLUT1 substrate that can be used as a probe for GLUT1 inhibitors. An accurate and simple assay for 2-NBDG in a HEK293T cell model overexpressing GLUT1 was developed using liquid chromatography-tandem mass spectrometry. Chromatographic separation was achieved using a Xbridge® Amide column (3.5 µm, 2.1 mm × 150 mm, Waters) with acetonitrile-water containing 2 µM ammonium acetate (80:20, v/v) at a flow rate of 0.25 mL/min. Mass detection was conducted in the parallel reaction monitoring (PRM) mode. The calibration curve for 2-NBDG showed good linearity in the concentration range of 5-500 ng/mL with satisfactory precision, a relative standard deviation ranging from 2.92 to 9.59% and accuracy with a relative error ranging from -13.14 to 7.34%. This method was successfully applied to quantify the uptake of GLUT1-mediated 2-NBDG, and the results clearly indicated inhibition of GLUT1 by WZB117 and quercetin (two potent glucose transporter inhibitors) in the GLUT1-HEK293T cell model. This study provides a convenient and accurate method for high-throughput screening of selective and promising GLUT1 inhibitors.

Sensitive Method for the Identification of Potential Sensitizing Impurities in Reaction Mixtures by Fluorescent Nitrobenzoxadiazole-Labeled Glutathione.

Allergic contact dermatitis is a critical issue in the development of new chemicals. Minor impurities with strong skin-sensitizing properties can be generated as byproducts. However, it is very difficult to identify these skin sensitizers in product mixtures. In this study, fluorescent nitrobenzoxadiazole-labeled glutathione (NBD-GSH) was synthesized to identify small amounts of skin sensitizers in reaction mixtures. Twelve known skin sensitizers and three nonsensitizers were reacted with NBD-GSH. Adducts formed only with the skin sensitizers, which allowed for their detection by a fluorescence detector. Liquid chromatography-mass spectrometry (LC-MS) analyses showed that NBD-GSH reacted with the skin sensitizers via its thiol and amino groups. An adduct of NBD-GSH with the strong skin sensitizer 1-chloro-2,4-dinitrobenzene was detected with a limit of detection of 6 × 10-8 mol/L by high-performance liquid chromatography with fluorescence detection. When a reaction mixture from primary alcohol oxidation was incubated with NBD-GSH, a NBD-GSH adduct formed with skin-sensitizing aldehyde impurities and could be specifically detected by LC-MS with fluorescence detection. This method will be useful for detection and identification of small amounts of skin sensitizers in raw materials, intermediates, reaction mixtures, and end products in the chemical industry.

Liver Plays a Major Role in FGF-21 Mediated Glucose Homeostasis.

BACKGROUND/AIMS: The liver is a vital organ in vertebrates and has a wide range of functions, including glucose absorption, glycogen storage and glucose production. Fibroblast growth factor (FGF)-21 is a metabolic regulator that is primarily produced by the liver. In this paper, we studied the effect of FGF-21 on glucose metabolism in the liver. METHODS: The glucose uptake of cells was detected by 2-Deoxy-d-[3H] glucose; the synergy between insulin and FGF-21 was evaluated. The mRNA expression of GLUT1-4, G6Pase and PEPCK was detected by real-time PCR. Glycogen synthesis was examined by the anthrone method. Blood samples to monitor glucose in db/db diabetic mice were obtained by tail snip. Glucose metabolism in the liver and adipose tissues was observed by fluorescence microscopy. RESULTS: In this study, FGF-21 stimulated glucose uptake by liver cells in both a dose and time-dependent manner, and at the same time, FGF-21 specifically stimulated GLUT1 expression in the liver cells. Furthermore, FGF-21 demonstrated a synergistic effect with insulin on glucose absorption, which is in accordance with enhanced GLUT-1 and -4 expression. Treatment with FGF-21 increased glycogen storage in liver cells. Consistent with in vitro results, FGF-21 lowered the plasma glucose level and stimulated GLUT1 expression and glycogen synthesis in db/db diabetic mice. Simultaneously, FGF-21 inhibited the gene expression of G6Pase and PEPCK. CONCLUSION: Our results suggest that FGF-21 clears up plasma glucose by stimulating glucose absorption in the liver of diabetic animals and decreases glucose release from the liver by inhibiting gluconeogenesis. Overall, these data indicate that the liver is an important target organ of FGF-21 to regulate glucose metabolism.

Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells.

Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C6-NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics.

Fluorescence probes to detect lipid-derived radicals.

Lipids and their metabolites are easily oxidized in chain reactions initiated by lipid radicals, forming lipid peroxidation products that include the electrophiles 4-hydroxynonenal and malondialdehyde. These markers can bind cellular macromolecules, causing inflammation, apoptosis and other damage. Methods to detect and neutralize the initiating radicals would provide insights into disease mechanisms and new therapeutic approaches. We describe the first high-sensitivity, specific fluorescence probe for lipid radicals, 2,2,6-trimethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)-6-pentylpiperidine-1-oxyl (NBD-Pen). NBD-Pen directly detected lipid radicals in living cells by turn-on fluorescence. In a rat model of hepatic carcinoma induced by diethylnitrosamine (DEN), NBD-Pen detected lipid radical generation within 1 h of DEN administration. The lipid radical scavenging moiety of NBD-Pen decreased inflammation, apoptosis and oxidative stress markers at 24 h after DEN, and liver tumor development at 12 weeks. Thus, we have developed a novel fluorescence probe that provides imaging information about lipid radical generation and potential therapeutic benefits in vivo.

Identification and Optimization of Combinatorial Glucose Metabolism Inhibitors in Hepatocellular Carcinomas.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. The expression of glucose transporter isoform 1, a key factor in transporting glucose into cancer cells, is overexpressed in several human cancers, including HCC. In addition, this has been shown to correlate with a higher proliferation index and more advanced stages in HCC, suggesting that inhibition of glucose metabolism is a promising therapeutic strategy. Our study used high-content screening (HCS) for compounds that target glucose metabolism and effect cell death in HCC cells. Specifically, we showed that a fluorescent 2-deoxyglucose analog, 2-[N-(7-nitrobenz-2- oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose, and CellTrace Calcein Red-Orange AM can be used reliably as readouts for glucose uptake and proliferative index, respectively, to identify drug candidates that simultaneously reduce glucose uptake and induce cell death in HCC cells. Thus, fluorescent glucose uptake bioprobes can be implemented in HCS assays to identify previously unknown regulators of glucose metabolism in HCC. In addition, our study also employs the use of feedback system control (FSC.II), a platform that optimizes the combinations of drugs identified through HCS. The coordinated use of HCS and FSC.II can improve the development of drug combinations and uncover previously unidentified signaling pathways that govern HCC as well as other cancers.

Vital-dye-enhanced multimodal imaging of neoplastic progression in a mouse model of oral carcinogenesis.

In this longitudinal study, a mouse model of 4-nitroquinoline 1-oxide chemically induced tongue carcinogenesis was used to assess the ability of optical imaging with exogenous and endogenous contrast to detect neoplastic lesions in a heterogeneous mucosal surface. Widefield autofluorescence and fluorescence images of intact 2-NBDG-stained and proflavine-stained tissues were acquired at multiple time points in the carcinogenesis process. Confocal fluorescence images of transverse fresh tissue slices from the same specimens were acquired to investigate how changes in tissue microarchitecture affect widefield fluorescence images of intact tissue. Widefield images were analyzed to develop and evaluate an algorithm to delineate areas of dysplasia and cancer. A classification algorithm for the presence of neoplasia based on the mean fluorescence intensity of 2-NBDG staining and the standard deviation of the fluorescence intensity of proflavine staining was found to separate moderate dysplasia, severe dysplasia, and cancer from non-neoplastic regions of interest with 91% sensitivity and specificity. Results suggest this combination of noninvasive optical imaging modalities can be used in vivo to discriminate non-neoplastic from neoplastic tissue in this model with the potential to translate this technology to the clinic.

HPLC-FLD determination of NBD-cholesterol, its ester and other metabolites in cellular lipid extracts.

22-[N(-7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-23,24-bisnor-5-cholen-3ß-ol (NBD-cholesterol), a fluorescent cholesterol analog, was an extragenous cholesterol tracer used to study cholesterol absorption and metabolism in cultured cells. In order to measure free intracellular cholesterol and its esters, a precise and sensitive method employing high-performance liquid chromatography/fluorescence detection (HPLC-FLD) was developed for the first time. Method validation showed a limit of detection at 30 ng/mL. The calibration curve was linear within the range of 0.0625-10.0 µg/mL (r(2) = 0.999). Accuracy and precision were highlighted by good recovery and low variations. Apart from NBD-cholesteryl oleate, two additional cellular metabolites of NBD-cholesterol, probably an isomer and an oxidation product, were determined in the lipid extracts of Caco-2 human colon adenocarcinoma cells according to mass spectrometry. In AC29 mouse malignant mesothelioma cells overexpressing acyl-CoA:cholesterol acyltransferase-1 (ACAT1) or ACAT2, only the oxidized metabolite was detected. Using the newly developed method, YIC-C8-434, a known ACAT inhibitor, was shown to inhibit ACAT activity in Caco-2 cells, as well as in AC29/ACAT1 or AC29/ACAT2 cells. In conclusion, the sensitive and specific HPLC-FLD method is a powerful tool for simultaneous quantification of intracellular NBD-cholesterol and its oleoyl-ester.

FLIM studies of 22- and 25-NBD-cholesterol in living HEK293 cells: plasma membrane change induced by cholesterol depletion.

HEK293 cells stably expressing δ-opioid receptor were labeled first with fluorescent analog of cholesterol, 22-NBD-cholesterol, exposed to cholesterol-depleting agent ß-cyclodextrin (ß-CDX) and analyzed by fluorescence lifetime imaging microscopy (FLIM). In accordance with chemical analysis of cholesterol level, the total cellular signal of this probe was decreased to half. Distribution of lifetime (τtot) values of 22-NBD-cholesterol, however, when screened over the whole cell area indicated no significant difference between control (τtot=4.9±0.1 ns) and ß-CDX-treated (τtot=4.8±0.1 ns) cells. On the contrary, comparison of control (τtot=5.1±0.1 ns) and ß-CDX-treated (τtot=4.4±0.1 ns) cells by analysis of 25-NBD-cholesterol fluorescence implied highly significant decrease of lifetime values of this probe. The observation that 22-NBD-cholesterol appears to be indifferent to the changes in the membrane packing in living cells is in agreement with previous studies in model membranes. However, our data indicate that the alternation of plasma membrane structure induced by decrease of cholesterol level by ß-CDX makes the membrane environment of NBD moiety of 25-NBD-cholesterol probe a significantly more hydrated. This finding not only encourages using 25-NBD-cholesterol in living cells, but also demonstrates that previously drawn discouraging conclusions on the use of 25-NBD-cholesterol in model membranes are not valid for living cells.

Resistance to a novel antichlamydial compound is mediated through mutations in Chlamydia trachomatis secY.

A novel and quantitative high-throughput screening approach was explored as a tool for the identification of novel compounds that inhibit chlamydial growth in mammalian cells. The assay is based on accumulation of a fluorescent marker by intracellular chlamydiae. Its utility was demonstrated by screening 42,000 chemically defined compounds against Chlamydia caviae GPIC. This analysis led to the identification of 40 primary-hit compounds. Five of these compounds were nontoxic to host cells and had similar activities against both C. caviae GPIC and Chlamydia trachomatis. The inhibitory activity of one of the compounds, (3-methoxyphenyl)-(4,4,7-trimethyl-4,5-dihydro-1H-[1,2]dithiolo[3,4-C]quinolin-1-ylidene)amine (MDQA), was chlamydia specific and was selected for further study. Selection for resistance to MDQA led to the generation of three independent resistant clones of C. trachomatis. Amino acid changes in SecY, a protein involved in Sec-dependent secretion in Gram-negative bacteria, were associated with the resistance phenotype. The amino acids changed in each of the resistant mutants are located in the predicted central channel of a SecY crystal structure, based on the known structure of Thermus thermophilus SecY. These experiments model a process that can be used for the discovery of antichlamydial, anti-intracellular, or antibacterial compounds and has led to the identification of compounds that may have utility in both antibiotic discovery and furthering our understanding of chlamydial biology.

Imaging of a glucose analog, calcium and NADH in neurons and astrocytes: dynamic responses to depolarization and sensitivity to pioglitazone.

Neuronal Ca(2+) dyshomeostasis associated with cognitive impairment and mediated by changes in several Ca(2+) sources has been seen in animal models of both aging and diabetes. In the periphery, dysregulation of intracellular Ca(2+) signals may contribute to the development of insulin resistance. In the brain, while it is well-established that type 2 diabetes mellitus is a risk factor for the development of dementia in the elderly, it is not clear whether Ca(2+) dysregulation might also affect insulin sensitivity and glucose utilization. Here we present a combination of imaging techniques testing the disappearance of the fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) as an indication of glycolytic activity in neurons and astrocytes. Our work shows that glucose utilization at rest is greater in neurons compared to astrocytes, and ceases upon activation in neurons with little change in astrocytes. Pretreatment of hippocampal cultures with pioglitazone, a drug used in the treatment of type 2 diabetes, significantly reduced glycolytic activity in neurons and enhanced it in astrocytes. This series of experiments, including Fura-2 and NADH imaging, provides results that are consistent with the idea that Ca(2+) levels may rapidly alter glycolytic activity, and that downstream events beyond Ca(2+) dysregulation with aging, may alter cellular metabolism in the brain.

Novel mouse model of colitis characterized by hapten-protein visualization.

Trinitrobenzene sulfonic acid (TNBS) and oxazolone are used to induce colitis for the investigation of inflammatory reactions in the colon. Although these chemicals are presumed to bind proteins in the colonic mucosa and then induce colitis as haptens, hapten-protein formation has not yet been confirmed in the colonic mucosa. We developed a mouse model of colitis characterized by hapten-protein visualization, using 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl), which emits fluorescence after binding to proteins. The enema of 1 mg/mL NBD-Cl induced severe diarrhea, rectal bleeding, and body weight reductions in BALB/c mice. Mucosal signs indicative of colitis, such as redness and swelling observed under stereomicroscopy or inflammatory cell infiltration and crypt-epithelium destruction under microscopy, were manifested around NBD-proteins visualized with fluorescence. Fluorescence microscopy showed the infiltration of F4/80+ cells around areas of NBD-proteins, and flow cytometry indicated the uptake of NBD-proteins by CD11b+ cells. We also found critical roles for T cells and interleukin-6 in colitis induction with NBD-proteins. NBD-Cl-induced colitis presents a unique model to study the relevance between hapten-protein formation and inflammatory reactions and offers a method to assess experimental interventions on colitis induction in the mucosa, where hapten-protein formation is confirmed.

Intracellular lipid droplets contain dynamic pools of sphingomyelin: ADRP binds phospholipids with high affinity.

During the last several years, intracellular lipid droplets have become the focus of intense study. No longer an inert bystander, the lipid droplet is now known as a dynamic organelle contributing lipids to many cellular events. However, while the dynamics of cholesterol efflux from both the plasma membrane and lipid droplets have been studied, less is known regarding the efflux of sphingomyelin from these membranes. In order to address this issue, sphingomyelin efflux kinetics and binding affinities from different intracellular pools were examined. When compared to the plasma membrane, lipid droplets had a smaller exchangeable sphingomyelin efflux pool and the time required to efflux that pool was significantly shorter. Fluorescence binding assays revealed that proteins in the plasma membrane and lipid droplet pool bound sphingomyelin with high affinity. Further characterization identified adipose differentiation-related protein (ADRP) as one of the sphingomyelin binding proteins in the lipid droplet fraction and revealed that ADRP demonstrated saturable binding to 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-hexanoyl)sphingosyl-phosphocholine (NBD-sphingomyelin) and also 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-phosphatidylcholine) with binding affinities in the nanomolar range. Taken together, these results suggest that lipid droplet associated proteins such as ADRP may play a significant role in regulating the intracellular distribution of phospholipids and lipids in general. Overall, insights from the present work suggest new and important roles for lipid droplets and ADRP in phospholipid metabolism.

New nonextractive and highly sensitive high-performance liquid chromatographic method for determination of paroxetine in plasma after offline precolumn derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.

New nonextractive and simple offline precolumn derivatization procedures have been proposed, for the first time, for the trace determination of paroxetine (PXT) in human plasma by HPLC with fluorescence detection. Trimetazidine (TMZ) was used as an internal standard. Plasma samples were treated with acetonitrile for protein precipitation and then derivatized with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole in borate buffer of pH 8 at 70 degrees C for 30 min. Separations of the derivatized PXT and TMZ were performed on a Nucleosil CN column using a mobile phase consisting of acetonitrile-10 mM sodium acetate buffer (pH 3.5)-methanol (47 + 47 + 6, v/v) at a flow rate of 1.0 mL/min. The derivatized samples were excited at 470 nm and monitored at an emission wavelength of 530 nm. Under the optimum chromatographic conditions, a linear relationship with good correlation coefficient (r = 0.9998, n = 7) was found between the peak area ratio and PXT concentrations in the range of 5-600 ng/mL. The LOD and LOQ were 1.37 and 4.14 ng/mL, respectively. The intraday and interassay precisions were satisfactory; the RSD did not exceed 4.2%. The accuracy of the method was proved by recovery of PXT from spiked human plasma at levels of 97.28-104.38 +/- 0.41-3.62%. The proposed method had high throughput, as the analysis involved a simple sample pretreatment procedure and short run time (< 10 min). The results demonstrated that the method would have a great value when it is applied in the therapeutic monitoring of PXT.

Utilization of fluorescence tracer in hyperinsulinemic-euglycemic clamp test in mice.

The hyperinsulinemic-euglycemic clamp test is considered to be a gold standard for the evaluation of insulin sensitivity. Here, a new version of the clamp test that used the fluorescence tracer 2-NBDG was tested. C57BL/6J mice were induced insulin resistant (IR) with a high-calorie diet. Rosiglitazone was administrated to IR mice and diabetic db/db mice. Insulin resistance was estimated with the oral glucose tolerance test (OGTT), the insulin tolerance test (ITT), the serum insulin level and the homeostasis model assessment of insulin resistance (HOMA-IR), and then confirmed by the hyperinsulinemic-euglycemic clamp test with 2-NBDG. The 2-NBDG content was measured by the fluorescence intensity. The characteristics of insulin resistance were shown remarkably with the increased values of serum insulin and HOMA-IR in IR mice, and with the results from OGTT and ITT in both IR and db/db mice. In the hyperinsulinemic-euglycemic clamp test, the glucose infusion rate and amount of 2-NBDG taken up into the liver, adipose, and skeletal muscle were decreased significantly in IR mice and db/db mice, respectively. The clearing rates of 2-NBDG from the circulation were much slower in both mouse models. All markers were reversed significantly by rosiglitazone treatment. The results indicate that with the fluorescence tracer 2-NBDG, the hyperinsulinemic-euglycemic clamp test can be used to estimate insulin sensitivity in vivo.

"Fluorescent glycogen" formation with sensibility for in vivo and in vitro detection.

There are presently many methods of detecting complex carbohydrates, and particularly glycogen. However most of them require radioisotopes or destruction of the tissue and hydrolysis of glycogen to glucose. Here we present a new method based on the incorporation of 2-NBDG (2-{N-[7-nitrobenz-2-oxa-1, 3-diazol 4-yl] amino}-2-deoxyglucose), a D-glucose fluorescent derivative, into glycogen. Two kinds of approaches were carried out by using Clone 9 rat hepatocytes as a cellular model; (1) Incubation of cell lysates with 2-NBDG, carbohydrate precipitation in filters and measurement of fluorescence in a microplate reader (2) Incubation of living hepatocytes with 2-NBDG and recording of fluorescence images by confocal microscopy. 2-NBDG labeled glycogen in both approaches. We confirmed this fact by comparison to the labeling obtained with a specific monoclonal anti-glycogen antibody. Also drugs that trigger glycogen synthesis or degradation induced an increase or decrease of fluorescence, respectively. This is a simple but efficient method of detecting glycogen with 2-NBDG. It could be used to record changes in glycogen stores in living cells and cell-free systems and opens the prospect of understanding the role of this important energy reserve under various physiological and pathophysiological conditions.

4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorogenic labeling reagent for the in vivo analysis of amino acid neurotransmitters using online microdialysis-capillary electrophoresis.

4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) has been characterized as a fluorogenic labeling reagent for high speed, online microdialysis-capillary electrophoresis (MD-CE) assays for amino acid neurotransmitters. NBD-F labeled amines are efficiently excited using 488 nm light allowing more common lasers to be used for excitation than has been previously possible. Amino acids were sampled using microdialysis, derivatized using NBD-F in an online reaction, and directly coupled to a high-speed CE separation with laser induced fluorescence (LIF) detection. Separation conditions were optimized allowing baseline separation of 16 amino acids, including glutamate, GABA, glycine, taurine, and D-serine, in 21.5 s. Parameters of the assay, including reaction temperature (80 degrees C), reaction time (5 min), NBD-F concentration (20 mM), and laser power (20 mW), were optimized. Limits of detection for glutamate and GABA were improved over previous assays by 4-fold and 25-fold, respectively. In vivo measurements were made in the rat striatum to demonstrate the effectiveness of the assay. Changes in in vivo concentrations as small as 8-9% could be observed for glycine, d-serine, and l-serine with statistical confidence. Changes in neurotransmitter concentrations induced by stimulation with high-K+ artificial cerebral spinal fluid (aCSF) were observed.

Fluorescent fructose derivatives for imaging breast cancer cells.

Breast cancer cells are known to overexpress Glut5, a sugar transporter responsible for the transfer of fructose across the cell membrane. Since Glut5 transporter is not significantly expressed in normal breast cells, fructose uptake can potentially be used to differentiate between normal and cancerous cells. Fructose was labeled with two fluorophores at the C-1 position: 7-nitro-1,2,3-benzadiazole (NBD) and Cy5.5. The labeling site was chosen on the basis of the presence and substrate specificity of the key proteins involved in the first steps of fructose metabolism. Using fluorescence microscopy, the uptake of the probes was studied in three breast cancer cell lines: MCF 7, MDA-MB-435, and MDA-MB-231. Both fluorescent fructose derivatives showed a very good uptake in all tested cell lines. The level of uptake was comparable to that of the corresponding glucose analogs, 2-NBDG and Cy5.5-DG. Significant uptake of 1-NBDF derivative was not observed in cells lacking Glut5 transporter, while the uptake of the 1-Cy5.5-DF derivative was independent of the presence of a fructose-specific transporter. While 1-NBDF showed Glut5-specific accumulation, the coupling of a large fluorophore such as Cy5.5 likely introduces big structural and electronic changes, leading to a fructose derivative that does not accurately describe the uptake of fructose in cells.

Rapid evaluation of the efficacy of microbial cell removal from fabrics.

The efficacy of microbial cell removal (EMR) from fabrics is a practically important indicator for the evaluation of cleansers and detergents. EMR is expressed quantitatively by the relative number of viable cells remaining on a fabric swatch after the treatment with these reagents. In order to count the viable cells on the swatch directly and rapidly, we have developed a unique microscopic imaging system with an ultra-deep focusing range. Standard swatches of cotton fabric were inoculated with microorganisms such as Pseudomonas fluorescence, Staphylococcus aureus, or Candida albicans. After the incubation on an agar medium, each swatch was treated with a fluorescent glucose, 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxyglucose, to stain only viable cells. The images of every cell distributed within the surface layer with no greater than 130 microm thickness could be integrated into one image. Thus visualized cells could be counted automatically by a novel imaging program. Using a pair of cotton swatches (0.5 x 1.0 cm(2)) inoculated with C. albicans, EMR was evaluated quantitatively. Before washing, the total number of viable cells found on the observation area (3.8 x 10(-4 )cm(2)) was 288 cells. After washing with a test detergent, no cell (<1) was detected. For this case, EMR was given by the formula: log(288/<1)=greater than 2.5. The imaging and cell count of a test fabric could be performed within 1 h.

Utilization of 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) for kinetic spectrophotometric assay of befunolol hydrochloride in its pharmaceutical formulation.

A simple, accurate, precise, and sensitive kinetic spectrophotometric method for determination of befunolol hydrochloride is described. The method is based on the formation of a colored product with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) in methanol at 70 degrees C for 45 min. The red-colored product was measured at 523 nm. The optimization of various experimental conditions is described; Beer's law was obeyed in the range 15.25 x 10(-6) to 122.04 x 10(-6) M. The results obtained showed good recoveries (100.1 +/- 0.80%). Application of the proposed method to a pharmaceutical formulation was successfully achieved. The determination of befunolol hydrochloride by fixed time, fixed concentration, and rate constant methods was feasible with the calibration equation obtained. However, the fixed time method proved to be more applicable.