Mikako Saito

A novel fluorescent derivative of glucose applicable to the assessment of glucose uptake activity of Escherichia coli

A novel fluorescent derivative of glucose was synthesized by reacting D-glucosamine and NBD-Cl. The TLC analysis of the reaction mixture showed the generation of a single spot with intense fluorescence (lambda Ex = 475 nm, lambda Em = 550 nm). The obtained novel fluorescent product, which was identified as 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) by 1H-NMR and FAB-MS spectrometries, was applied to the assessment of the glucose uptake activity of Escherichia coli B. 2-NBDG accumulated in living cells and not in dead cells. The uptake of 2-NBDG was competitively inhibited by D-glucose and not by L-glucose, which suggested the involvement of the glucose transporting system in the uptake of 2-NBDG. 2-NBDG taken into the cytoplasma of E. coli cells was supposedly converted into another derivative in the glucose metabolic pathway.

Measurement of glucose uptake and intracellular calcium concentration in single, living pancreatic beta-cells.

There has been no method previously to measure both glucose transport and its effect on the various intracellular functions in single, living mammalian cells. A fluorescent derivative of d-glucose, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG), that we have developed has made such measurements possible. COS-1 cells that overexpress the human glucose transporter GLUT2 show significantly greater 2-NBDG uptake than mock transfected cells. Using GLUT2-abundant mouse insulin-secreting clonal MIN6 cells, we found that 2-NBDG was incorporated into the cells in a time- and concentration-dependent manner. The 2-NBDG uptake was inhibited by high concentrations of d-glucose in a dose-dependent manner and also was almost completely inhibited by 10 μm cytochalasin B. We then measured both glucose uptake and the intracellular calcium concentration ([Ca2+] i ) in single, living pancreatic islet cells. 2-NBDG and fura-2 were used as the tracer of glucose and indicator of intracellular calcium, respectively. All of the cells that showed an increase in [Ca2+] i in response to a high concentration of glucose (16.8 mm) rapidly incorporated significant 2-NBDG. Immunocytochemical examination confirmed these cells to be insulin-positive β-cells. All of the cells that showed no significant, rapid 2-NBDG uptake lacked such glucose responsiveness of [Ca2+] i , indicating that these cells were non-β-cells such as glucagon-positive α-cells. These results show the uptake of glucose causing a concomitant increase of [Ca2+] i in β-cells. Because 2-NBDG is incorporated into mammalian cells through glucose transporters, it should be useful for the measurement of glucose uptake together with concomitant intracellular activities in many types of single, living mammalian cells.

A real-time method of imaging glucose uptake in single, living mammalian cells

This protocol details a method for monitoring glucose uptake into single, living mammalian cells using a fluorescent D-glucose derivative, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), as a tracer. The specifically designed chamber and superfusion system for evaluating 2-NBDG uptake into cells in real time can be combined with other fluorescent methods such as Ca2+ imaging and the subsequent immunofluorescent classification of cells exhibiting divergent 2-NBDG uptake. The whole protocol, including immunocytochemistry, can be completed within 2 d (except for cell culture). The procedure for 2-NBDG synthesis is also presented.

A novel glucosylation reaction on anthocyanins catalyzed by acyl-glucose-dependent glucosyltransferase in the petals of carnation and delphinium.

This work describes a glucosylation reaction at the 5/7 positions of anthocyanins in the petals of carnations and delphiniums. Unusually, this reaction is catalyzed by acyl-glucose–dependent glucosyltransferases that belong to glycoside hydrolase family 1. This modification mechanism may play an important role in generating variation in anthocyanins. Glucosylation of anthocyanin in carnations (Dianthus caryophyllus) and delphiniums (Delphinium grandiflorum) involves novel sugar donors, aromatic acyl-glucoses, in a reaction catalyzed by the enzymes acyl-glucose–dependent anthocyanin 5(7)-O-glucosyltransferase (AA5GT and AA7GT). The AA5GT enzyme was purified from carnation petals, and cDNAs encoding carnation Dc AA5GT and the delphinium homolog Dg AA7GT were isolated. Recombinant Dc AA5GT and Dg AA7GT proteins showed AA5GT and AA7GT activities in vitro. Although expression of Dc AA5GT in developing carnation petals was highest at early stages, AA5GT activity and anthocyanin accumulation continued to increase during later stages. Neither Dc AA5GT expression nor AA5GT activity was observed in the petals of mutant carnations; these petals accumulated anthocyanin lacking the glucosyl moiety at the 5 position. Transient expression of Dc AA5GT in petal cells of mutant carnations is expected to result in the transfer of a glucose moiety to the 5 position of anthocyanin. The amino acid sequences of Dc AA5GT and Dg AA7GT showed high similarity to glycoside hydrolase family 1 proteins, which typically act as β-glycosidases. A phylogenetic analysis of the amino acid sequences suggested that other plant species are likely to have similar acyl-glucose–dependent glucosyltransferases.

Evaluation of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose, a new fluorescent derivative of glucose, for viability assessment of yeast Candida albicans

A new fluorescent derivative of d-glucose, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG), which had been previously developed for the analysis of glucose uptake activity by living cells, was investigated to evaluate its applicability for assaying the viability of yeast Candida albicans. Lineweaver-Burk plots showed the uptake of 2-NBDG to be competitively inhibited by d-glucose and not by l-glucose, which suggested the involvement of the glucose transporting system of C. albicans in the uptake of 2-NBDG. A good correlation was obtained between the yeast viability, determined by the plate-count method, and the 2-NBDG uptake activity of yeast cells (correlation constant: r=0.97). This is expected to lead to the development of a new fluorescent probe for the determination of yeast cell viability.

Novel β‐1,3‐, 1,6‐oligoglucan elicitor from Alternaria alternata 102 for defense responses in tobacco

A novel elicitor that induces chitinases in tobacco BY‐2 cells was isolated from Alternaria alternata 102. Six other fungi, including A. alternata IFO 6587, could not induce, or weakly induce chitinase activity. The purified elicitor was soluble in 75% methanol and showed the chitinase‐inducing activity when applied at concentrations of as low as 25 ng·mL−1. Structural determination by methylation analysis, reducing‐end analysis, MALDI‐TOF/MS, and NMR spectroscopy indicated that the elicitor was a mixture of β‐1,3‐, 1,6‐oligoglucans mostly with a degree of polymerization of between 8 and 17. Periodate oxidation of the elicitor suggested that the 1,6‐linked and nonreducing terminal residues are essential for the elicitor activity. Further analysis of the elicitor responses in BY‐2 cells indicated that the activity of this β‐1,3‐, 1,6‐glucan elicitor was about 1000 times more potent than that of laminarin, which is a known elicitor of defense responses in tobacco. Analyzing the expression of defense‐related genes indicated that a phenylalanine ammonia‐lyase gene and a coumaroyl‐CoA O‐methyltransferase gene were transiently expressed by this β‐1,3‐, 1,6‐glucan elicitor. The elicitor induced a weak oxidative burst but did not induce cell death in the BY‐2 cells. In the tissue of tobacco plants, this β‐1,3‐, 1,6‐glucan elicitor induced the expression of basic PR‐3 genes, the phenylpropanoid pathway genes, and the sesquiterpenoid pathway genes. In comparison with laminarin and laminarin sulfate, which are reported to be potent elicitors of defense responses in tobacco, the expression pattern of genes induced by the purified β‐1,3‐, 1,6‐glucan elicitor was more similar to that induced by laminarin than to that induced by laminarin sulfate.

Single-cell viability assessment with a novel spectro-imaging system.

Single-cell viability assessment by means of plural dye probes require the spectral and temporal analysis of microscopic images of the test cells. To meet this requirement, we have developed a simple and compact spectro-imaging system using an image slicer and a grism. The image slicer was made of a bundle of 100 optical fibers. The field of view is divided into 10 x 10 sections. The spectral data of each section could be recorded every 5 s in the range from 400 to 800 nm at 5 nm resolution. The viability changes of yeast or tobacco single-cells were measured with this system. Using BY-2 cells, for example, the response to a chemical stress of saponin was measured by means of two fluorescent probes. The spectral-spatial-temporal data of fluorescein and DNA bound ethidium bromide provided us with useful information about the dynamic change of cell membrane permeability from which the cell viability was assessed.

Isolation and Properties of an Extracellular β-Glucosidase from a Filamentous Fungus, Cladosporium resinae, Isolated from Kerosene

An extracellular β-glucosidase was purified from a culture filtrate of the fungus Cladosporium resinae strain NK-1 grown on a medium containing starch, Tween 80, and yeast extract. The purified enzyme was monomeric with an Mr 98,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native gel filtration using HPLC. The enzyme had optimal activity with p-nitrophenyl-β-D-glucoside (PNPG) at 50°C and pH 4.5. The enzyme catalyzed the hydrolysis of cellobiose and PNPG. The Km and Vmax with PNPG as the substrate at 50°C and pH 4.5 were 0.07 mM and 364 μmol/min/mg, respectively; with cellobiose as the substrate, the corresponding values were 2.3 mM and 75 μmol/min/mg. The enzyme activity was competitively inhibited by glucose (Ki=20 mM), while fructose, galactose, mannose, arabinose, xylose (each at 50 mM), sucrose, and lactose (each at 30 mM) were not inhibitory. While the enzyme has activity against sophorose (β-1,2-glucobiose) and laminaribiose (β-1,3-glucobiose), it has no activity against gentiobiose (β-1,6-glucobiose). The activity of the β-glucosidase was inhibited by Ag(+), Fe(2+), Mn(2+), Zn(2+), Hg(2+), SDS, and p-chloromercuribenzoate.

Noise-free accurate count of microbial colonies by time-lapse shadow image analysis

Microbial colonies in food matrices could be counted accurately by a novel noise-free method based on time-lapse shadow image analysis. An agar plate containing many clusters of microbial colonies and/or meat fragments was trans-illuminated to project their 2-dimensional (2D) shadow images on a color CCD camera. The 2D shadow images of every cluster distributed within a 3-mm thick agar layer were captured in focus simultaneously by means of a multiple focusing system, and were then converted to 3-dimensional (3D) shadow images. By time-lapse analysis of the 3D shadow images, it was determined whether each cluster comprised single or multiple colonies or a meat fragment. The analytical precision was high enough to be able to distinguish a microbial colony from a meat fragment, to recognize an oval image as two colonies contacting each other, and to detect microbial colonies hidden under a food fragment. The detection of hidden colonies is its outstanding performance in comparison with other systems. The present system attained accuracy for counting fewer than 5 colonies and is therefore of practical importance.

Viable Cell Detection by the Combined Use of Fluorescent Glucose and Fluorescent Glycine

The combined use of a fluorescent glucose (2NBDG) and a fluorescent glycine (NBD-Gly) was tried for the detection of viable cells of significant foodborne pathogenic strains in addition to several Escherichia coli strains and coliforms. Thirty-five out of 41 strains showed marked uptake of 2NBDG but 6 strains were not able to take in 2NBDG. Five out of these 6 strains showed NBD-Gly uptake.