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.     

Asymmetric transport of a fluorescent glucose analogue by human erythrocytes

A fluorescent glucose analogue, 6-deoxy-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-aminoglucose (NBDG), was synthesized and its interactions with the hexose transport system of the human red blood cell were investigated. NBDG entry is inhibited by increasing concentrations of d-glucose (K_i = 2 mM). However, NBDG exit is unaffected by d-glucose in red blood cells. Cytochalasin B was found to inhibit both NBDG entry and exit. NBDG accumulates in the red blood cell above the theoretical equilibrium concentration. Accumulation of NBDG is temperature-sensitive and is due to the binding of NBDG to some intracellular substance. The binding of NBDG to purified hemoglobin suggests that accumulation of NBDG by erythrocytes is due to the intracellular binding of NBDG to hemoglobin. NBDG does not accumulate in pink erythrocyte ghosts, while its rate of uptake is still inhibited by d-glucose and cytochalasin B. Although there was no apparent d-glucose inhibition of NBDG exit by intact red blood cells, d-glucose was able to inhibit NBDG exit by pink erythrocyte ghosts. The differing properties of NBDG influx and efflux support the interpretation that the hexose transport system of the human red blood cell appears asymmetric although it may be intrinsically symmetric.     

Glucose metabolism down‐regulates the uptake of 6‐(N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)‐2‐deoxyglucose (6‐NBDG) mediated by glucose transporter 1 isoform (GLUT1): theory and simulations using the symmetric four‐state carrier model

The non‐metabolizable fluorescent glucose analogue 6‐(N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)‐2‐deoxyglucose (6‐NBDG) is increasingly used to study cellular transport of glucose. Intracellular accumulation of exogenously applied 6‐NBDG is assumed to reflect concurrent gradient‐driven glucose uptake by glucose transporters (GLUTs). Here, theoretical considerations are provided that put this assumption into question. In particular, depending on the microscopic parameters of the carrier proteins, theory proves that changes in glucose transport can be accompanied by opposite changes in flow of 6‐NBDG. Simulations were carried out applying the symmetric four‐state carrier model on the GLUT1 isoform, which is the only isoform whose kinetic parameters are presently available. Results show that cellular 6‐NBDG uptake decreases with increasing rate of glucose utilization under core‐model conditions, supported by literature, namely where the transporter is assumed to work in regime of slow reorientation of the free‐carrier compared with the ligand–carrier complex. To observe an increase of 6‐NBDG uptake with increasing rate of glucose utilization, and thus interpret 6‐NBDG increase as surrogate of glucose uptake, the transporter must be assumed to operate in regime of slow ligand–carrier binding, a condition that is currently not supported by literature. Our findings suggest that the interpretation of data obtained with NBDG derivatives is presently ambiguous and should be cautious because the underlying transport kinetics are not adequately established.     

A fluorescent compound for glucose uptake measurements in isolated rat cardiomyocytes

A focus of current diabetes research is the development of insulinomimetic compounds for oral treatment of diabetes and its associated cardiac complications. Screening compounds for their potential insulinomimetic effects usually involves the use of radioactive isotopes. The focus of this study was to investigate a nonradioactive fluorescent compound for its use in screening insulinomimetic compounds. The indicator 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) has been used by some workers to measure glucose uptake in Escherichia coli and Candida albicans. We propose that 2-NBDG will also be a suitable indicator for mammalian cell lines, in particular rat cardiomyocytes. We found that the indicator could give a reliable reproducible standard curve following appropriate dilution and is taken up by isolated cardiomyocytes. The insulinomimetic compounds vanadyl sulfate and sodium molybdate showed rates of glucose uptake similar to that of insulin. Furthermore, the rate of uptake measured for insulin using this technique (0.04 +/- 0.003 nmol x min(-1) x 10(6) cells(-1) is comparable with previous literature using 2-deoxyglucose uptake measurements on isolated myocytes (0.040 nmol x min(-1) x 10(6) cells(-1), demonstrating the validity of this fluorescent compound for glucose uptake studies.     

Regional differences in glucose transport in the mouse hippocampus

Summary In order to observe glucose transport into the brain, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (NBDG), a non-metabolizable and fluorescent glucose analogue, was injected intravenously into mice. After ascertaining that this glucose analogue is non-metabolizable in the brain, the NBDG contents in the blood and brain were measured quantitatively by spectrofluorimetry at 0, 0.5, 2, 5, 10 and 30 min after intravenous injection. The NBDG content in the blood decreased markedly with time, whereas in the brain it rapidly decreased, then gradually increased after 2 min. Glucose transport into the hippocampus was observed with a confocal laser scanning microscope. At 0.5 min, NBGD was seen to be highly concentrated on the vascular wall. Using the confocal mode, it was found that the fluorescence was unevenly distributed on the microvessel wall, suggesting local differences of glucose transport in the vascular wall. At 5 min, the fluoresent intensity of the vascular wall was markedly decreased, whereas relatively intense fluorescence was observed in the cerebral parenchyma of the stratum lacunosum-moleculare and stratum pyramidale of CA3. At 10 min, a weak fluoresence was diffusely distributed in the hippocampus. As to the localization of NBDG in the brain, capillary endothelium (luminal and abluminal membrane), basement membrane, and the feet of the astrocytes are discussed.     

Characterization and evaluation of two novel fluorescent sigma-2 receptor ligands as proliferation probes

We synthesized and characterized two novel fluorescent sigma-2 receptor selective ligands, SW120 and SW116, and evaluated these ligands as potential probes for imaging cell proliferation. Both ligands are highly selective for sigma-2 receptors versus sigma-1 receptors. SW120 and SW116 were internalized into MDA-MB-435 cells, and 50% of the maximum fluorescent intensity was reached in 11 and 24 minutes, respectively. In vitro studies showed that 50% of SW120 or SW116 washed out of cells in 1 hour. The internalization of SW120 was reduced ≈30% by phenylarsine oxide, an inhibitor of endocytosis, suggesting that sigma-2 ligands are internalized, in part, by an endocytotic pathway. Subcellular localization studies using confocal and two-photon microscopy showed that SW120 and SW116 partially colocalized with fluorescent markers of mitochondria, endoplasmic reticulum, lysosomes, and the plasma membrane, suggesting that sigma-2 receptors localized to the cytoplasmic organelles and plasma membrane. SW120 did not colocalize with the nuclear dye 4',6-diamidino-2-phenylindole. In vivo studies showed that the uptake of SW120 in solid tumors and peripheral blood mononuclear cells of mice positively correlated with the expression level of the cell proliferation marker Ki-67, suggesting that sigma-2 fluorescent probes may be used to image cell proliferation in mice.     

A Chinese hamster ovary cell mutant defective in the non-endocytic uptake of fluorescent analogs of phosphatidylserine: isolation using a cytosol acidification protocol

Transmembrane movement of phosphatidylserine (PS) and various PS analogs at the plasma membrane is thought to occur by an ATP-dependent, protein-mediated process. To isolate mutant CHO cells defective in this activity, we first obtained conditions which inhibited the endocytic, but not the non-endocytic pathway of lipid internalization since PS may enter cells by a combination of these two pathways. We found that acidic treatment of cells, which blocks clathrin-dependent endocytosis, enhanced the energy-dependent uptake of 1-palmitoyl-2-(6-[(7-nitrobenz- 2-oxa-1,3-diazol-4-yl)amino]caproyl -sn- glycero-3-phosphoserine (C6- NBD-PS) in CHO cells from donor vesicles (liposomes) by about twofold. Control experiments demonstrated that the enhanced uptake of C6-NBD-PS at acidic pH was not due to: (a) an increase in the capacity of the plasma membrane to incorporate C6-NBD-PS from the donor vesicles; (b) a decrease in the rate of loss of C6-NBD-PS from the cells; or (c) fusion or engulfment of the donor vesicles. When cytosolic acidification (to pH 6.3) was imposed without acidification of the extracellular medium, C6-NBD-PS uptake by intact cells was increased by about 50% compared to control values determined in the absence of acidification. These results suggested that a protein and energy dependent system(s) for transbilayer movement of the fluorescent PS was stimulated by cytosolic acidification. A screening method for mutant cells defective in the non- endocytic uptake of fluorescent PS analogs with replica cell colonies at acidic pH was then devised. After selection of mutagenized CHO-K1 cells by in situ screening, we obtained a mutant cell line in which uptake of fluorescent PS analogs was reduced to about 25% of the wild type level at either pH 6.0 or 7.4. Control experiments demonstrated that the reduced uptake of fluorescent PS analogs in the mutant cells was unrelated to multidrug resistance, and that endocytosis of another plasma membrane lipid marker occurred normally in the mutant cells. These results suggested that a non-endocytic pathway responsible for uptake of fluorescent PS analogs was specifically affected in the mutant cells.     

Cytofluorometric analysis for estrogen receptors using fluorescent estrogen probes

Estrogen receptor (ER) analysis of breast cancer tissue has been shown to be very useful in predicting which patients will respond to hormone therapy and have a better prognosis. The ER assay is, however, tedious and time consuming. Measurement of ER by flow cytometry would be rapid and based on either an average fluorescence-E2 probe intensity per cell or the percentage of the ER+ cells per cell suspension. Analysis of E2 modified structures for relative binding affinity to the ER determined by competition studies and for fluorescence uptake into cell suspensions determined by flow cytometry was performed. Lack of high affinity to the ER and purity of the compound were major problems for the fluorescein-labeled estrogen probes. Base hydrolysis of the ester linkage in fluorescein-E2 compounds demonstrated by HPLC very little estradiol derivative in the parent compounds compared to total components present. A second type of fluoresceinated estrogen which has a peptide bond between the steroid and the chromophore was also tested. It was less contaminated but was unable to get into the cell and showed no binding activity to the ER. A pure plant fluorescent estrogen, coumestrol, has Ka of 6 X 10(8) M-1 for the ER and is a single component as determined by HPLC. Specific fluorescent uptake of coumestrol was performed on ER+ and ER- viable cell suspensions. When these coumestrol-cell suspensions were excited at 350-360 nm and the blue emission was measured using flow cytometry, the result was a fluorescence uptake that was not highly displaceable by excess nonfluorescence E2 probes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity of pyoverdine-mediated iron uptake among fluorescent Pseudomonas strains.

Pyoverdine-mediated iron transport was determined for seven fluorescent Pseudomonas strains belonging to different species. For all strains, cell or cell outer membrane and iron(III)-pyoverdine combinations were compared with their homologous counterparts in uptake, binding, and cross-feeding experiments. For four strains (Pseudomonas putida ATCC 12633, Pseudomonas fluorescens W, P. fluorescens ATCC 17400, and Pseudomonas tolaasii NCPPB 2192), the pyoverdine-mediated iron transport appeared to be strictly strain specific; pyoverdine-facilitated iron uptake by iron-starved cells and binding of ferripyoverdine to the purified outer membranes of such cells were efficient only in the case of the homologous systems. Cross-feeding assays, in liquid or solid cultures, resulted, however, especially for P. fluorescens ATCC 17400, in some discrepancies compared with uptake and binding assays, suggesting that growth experiments are the least likely to yield correct information on specificity of the pyoverdine-mediated iron transport. For the three other strains (P. fluorescens ATCC 13525, P. chlororaphis ATCC 9446, and P. aeruginosa ATCC 15692), cross-reactivity was demonstrated by the uptake, binding, and cross-feeding experiments. In an attempt to determine which parts of the iron transport system were responsible for the specificity, the differences in amino acid composition of the pyoverdines, together with the differences observed at the level of the iron-sensitive outer membrane protein pattern of the seven strains, are discussed.     

Intrinsically fluorescent PAMAM dendrimer as gene carrier and nanoprobe for nucleic acids delivery: bioimaging and transfection study

This study successfully evaluated gene delivery and transfection toward rat C6 glioma cell lines mediated by intrinsic blue fluorescent poly(amido amine) (PAMAM) dendrimer. We used three antisense oligonucleotides, (AS-ODN) p75, NGF1, and NGF2 for knocking down specific protein expressions. The three oligonucleotides were electrostatically associated with the photoluminescent amino-terminated PAMAM dendrimer to yield fluorescent complexes at various nitrogen-to-phosphorus (N/P) ratios. Compared with pristine PAMAM dendrimer and hyperbranched polyethylenimine (PEI), the fluorescent PAMAM dendrimer revealed lower in vitro cytotoxicity toward C6 cells, allowing us to transfect the cells with the AS-ODN complexes under a higher N/P ratio. Due to the intrinsic fluorescence, cellular uptake behavior could be directly analyzed by fluorescence microscopy and flow cytometry, without additional fluorescence labeling. As expected, the result clearly suggested that the uptake efficiency increased as the N/P value increased. Furthermore, the quantified data obtained from flow cytometry indicated relatively higher uptake efficiency for the p75 complex, which is mainly due to different association patterns between the fluorescent dendrimer and AS-ODNs. At N/P = 20, atomic force microscopic analysis confirmed that the p75 complex formed well-condensed, spherical particles with dimensions less than 200 nm, but that NGF2 AS-ODN associated poorly with the dendrimer. Finally, Western blot analysis indicated that these complexes were capable of knocking down the specific protein expression to a certain level, being comparable to the hyperbranched PEI-mediated gene transfection. Our preliminary results clearly indicated that intrinsic fluorescent PAMAM dendrimers show promise as gene vehicles that can achieve delivery, transfection, and bioimaging at the same time.     

Interplay between the efflux pump and the outer membrane permeability barrier in fluorescent dye accumulation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa encodes three types of xenobiotic efflux pumps, MexAB-OprM, MexCD-OprJ, and MexEF-OprN, which are regulated by the nalB, nfxB, and nfxC genes, respectively, and their high expression renders the cells resistant to multiple species of antibiotics. We evaluated the role of the outer membrane permeability barrier and the efflux pump in lowering the intracellular concentration of fluorescent probes. The wild-type, nalB, nfxB, and nfxC strains with an intact outer membrane showed equally high capability in draining out intracellular fluorescent dye, 2-(4-dimethylaminostyryl)-1-ethylpyridinium and ethidium bromide. When the outer membrane barrier was dismantled by the EDTA treatment, wild-type, nfxC, nfxB, and nalB strains showed significantly different levels of dye accumulation. The polymyxin B-treated cells showed an even more pronounced difference in dye accumulation among the nfxC, nfxB, and nalB mutants. We concluded from these results that the xenobiotic extrusion pumps interplay with the outer membrane permeability barrier in lowering the intracellular substrate concentration. Among three extrusion pumps in P. aeruginosa, MexAB-OprM was the most efficient, followed by MexCD-OprJ and MexEF-OprN pumps for the fluorescent dye extrusion.     

Dual probe with fluorescent and magnetic properties for imaging solid tumor xenografts

A dual probe with fluorescent and magnetic reporter groups was constructed by linkage of the near-infrared (NIR) fluorescent transferrin conjugate (Tf(NIR)) on the surface of contrast agent-encapsulated cationic liposome (Lip-CA). This probe was used for magnetic resonance imaging (MRI) and optical imaging of MDA-MB-231-luc breast cancer cells grown as a monolayer in vitro and as solid tumor xenografts in nude mice. Confocal microscopy, optical imaging, and MRI showed a dramatic increase of in vitro cellular uptake of the fluorescent and magnetic reporter groups from the probe compared with the uptake of contrast agent or Lip-CA alone. Pretreatment with transferrin (Tf) blocked uptake of the probe reporters, indicating the importance and specificity of the Tf moiety for targeting. Intravenous administration of the dual probe to nude mice significantly enhanced the tumor contrast in MRI, and preferential accumulation of the fluorescent signal was clearly seen in NIR-based optical images. More interestingly, the contrast enhancement in MRI showed a heterogeneous pattern within tumors, which reflected the tumor's morphologic heterogeneity. These results indicate that the newly developed dual probe enhances the tumor image contrast and is superior to contrast agent alone for identifying the tumor pathologic features on the basis of MRI but also is suitable for NIR-based optical imaging.     

Vibrio azureus emits blue-shifted light via an accessory blue fluorescent protein

Luminous marine bacteria usually emit bluish-green light with a peak emission wavelength (λ(max) ) at about 490?nm. Some species belonging to the genus Photobacterium are exceptions, producing an accessory blue fluorescent protein (lumazine protein: LumP) that causes a blue shift, from λ(max) ≈?490 to λ(max) ≈?476?nm. However, the incidence of blue-shifted light emission or the presence of accessory fluorescent proteins in bacteria of the genus Vibrio has never been reported. From our spectral analysis of light emitted by 16 luminous strains of the genus Vibrio, it was revealed that most strains of Vibrio azureus emit a blue-shifted light with a peak at approximately 472?nm, whereas other Vibrio strains emit light with a peak at around 482?nm. Therefore, we investigated the mechanism underlying this blue shift in V.?azureus NBRC 104587(T) . Here, we describe the blue-shifted light emission spectra and the isolation of a blue fluorescent protein. Intracellular protein analyses showed that this strain had a blue fluorescent protein (that we termed VA-BFP), the fluorescent spectrum of which was almost identical to that of the in vivo light emission spectrum of the strain. This result strongly suggested that VA-BFP was responsible for the blue-shifted light emission of V.?azureus.     

Evaluation of glucose transport and its regulation by insulin in human monocytes using flow cytometry.

BACKGROUND: We investigated the effects of insulin on glucose transport in human monocytes using flow cytometry, a method with several advantages over previously used techniques. We hypothesized that monocytes could be used as tools to study insulin action at the cellular level and facilitate the investigation of mechanisms that lead to insulin resistance. METHODS: Blood was withdrawn from 38 healthy subjects. The expression of glucose transporter (GLUT) isoforms in plasma membrane and the rates of glucose transport were determined with and without insulin (10 to 1,000 mU/L). Anti-CD14 phycoerythrin monoclonal antibody was used for monocyte gating. GLUT isoforms were determined after staining cells with specific antisera to GLUT1, GLUT3, and GLUT4. Glucose transport was monitored with 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (NBDG). RESULTS: Insulin increased the uptake of NBDG (median effective dose 20 mU/L) and the expression of GLUT3 and GLUT4 isoforms in the plasma membrane (median effective doses 20 and 35 mU/L, respectively) but had no effect on GLUT1. Maximal effects were always reached at 100 mU/L of insulin. CONCLUSIONS: Monocytes may be a valid model system to study the effects of insulin on glucose transport. Further, flow cytometry is suitable for this investigation and can be used as an alternative to radiotracer methods.     

New fluorescent cytidine 5''-phosphate derivatives.

Interaction of cytidine 5'-phosphate with chloroacetone or p-tosyloxyacetone leads to 2-methyl-5,6-dihydro-5-oxo-6-(5-0-phospho-beta-D-ribofuranosyl)-imidazo/1,2-c/pyrimidine (2-methylethenocytidine 5'-phosphate) whereas analogous reaction with phenacyl bromide produces similar 2-phenyl-derivative. The bicyclic nucleotides obtained showed significant UV absorption at long wavelength where common nucleotides and proteins exhibited no absorption. These derivatives are highly fluorescent when heterocyclic ring is protonated. The absorption and fluorescent properties of the substituted ethenocytidine 5'-phosphoate derivatives seem to be suitable for their use as fluorescent probes or labels in biochemical studies.     

Growth promotion and inhibition by antibiotic-producing fluorescent pseudomonads on citrus roots

Summary Forty-three strains of feeder root colonizing fluorescent pseudomonads from rough lemon (Citrus jambhiri Lush.) roots were examined for effects on rough lemon and sweet orange (Citrus sinensis Osbeck) seedlings. Plants inoculated with a single bacterial soil-drench had, after 10 months, a range of stimulatory (to 116%) and inhibitory effects (to 52%). Stimulatory bacteria particularly increased growth of root systems. Cultivar-specific inhibition and stimulation was evident in inoculations of rough lemon and sweet orange seedlings. Populations of fluorescent rhizobacteria on inoculated and noninoculated, as well as on stimulated and nonstimulated seedlings, did not differ significantly (10.8×10⁶ to 30.3×10⁶ CFU/g root). Population of fluorescent rhizobacteria on seedlings were higher than populations on feeder roots from grove trees (2.8 to 5.7×10⁶ CFU/g). Ninety-four and 81% of 251 fluorescent strains produced antibiotics against the fungusGeotrichum candidum and the bacteriumErwinia stewartii, respectively. Antibiotic activities of 90% of the antibiotic producing strains were repressed by Fe³⁺, indicating siderophore production. In comparison, only 9.6 and 15% of 94 randomly selected nonfluorescentPseudomonas strains were antibiotic producers. Differences between stimulatory and inhibitory or neutral bacteria were not apparent from antibiosis tests. On the basis of physiological tests,Pseudomonas putida was the most abundant (>62%) pseudomonad species on rough lemon roots. Growth stimulating strains appeared to be in bothP. putida andP. fluorescens groups. FewP. aeruginosa strains were identified on citrus roots.Florida Agricultural Experiment Stations Journal Series No.     

The widespread occurrence of rutin glycosidase in fluorescent phytopathogenic pseudomonads

Two hundred and twelve strains representing 11 species of fluorescent pseudomonads along with an additional 198 strains isolated from the leaf and root surfaces of plants were tested for rutin glycosidase, on a mineral base medium supplemented with rutin and glucose. AH of the arginine dihydrolase negative phytopathogens (182 strains) with the exception of 12 of 12 strains of Pseudomonas syringae pv. glycina and one of 28 strains of P. syringae pv. phaseoticola showed rutin glycosidase activity whereas few of the arginine dihydrolase positive strains (22 of 228) were positive.     

Novel use of fluorescent glucose analogues to identify a new class of triazine-based insulin mimetics possessing useful secondary effects

There is an urgent need to discover new compounds that effectively treat diabetes by mimicking the action of insulin (insulin mimetics). Traditional approaches to studying anti-diabetic agents in cells are inconvenient for screening chemical libraries to identify insulin mimetics. 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) and 6-NBDG are fluorescent analogues of glucose that could be employed in screening. However, there are no published data about the use of these analogues to identify new insulin mimetics. We have developed a screening system based on 6-NBDG using 3T3-L1 adipocytes in a 96-well culture plate format. 6-NBDG was found to produce a larger signal than 2-NBDG in this screening system. 6-NBDG uptake in 3T3-L1 adipocytes was sensitive to insulin, known insulin mimetics, inhibitors of glucose transport and insulin-sensitizing compounds. To validate our screening system, a chemical library of 576 tagged, triazine-based small molecules was screened. The screening results were identical to that obtained from a commercial enzyme-based glucose assay. Two inducers of glucose uptake were shown to be non-cytotoxic and confirmed as insulin mimetic compounds by their inhibition of epinephrine-stimulated free fatty acid release from adipocytes. These novel insulin mimetics functioned at a markedly lower concentration than two widely studied insulin mimetics, zinc(ii) complexes and vanadium compounds, and also showed novel, beneficial effects on endothelial cell function (a key determinant of secondary complications in diabetes). The discovery of new insulin mimetics using 6-NBDG validates the use of this probe in the development of large-scale, cell-based screening systems based on the uptake of fluorescent-tagged glucose analogues. This research should aid the development of novel strategies to discover new drugs and drug targets for combating the increasing prevalence of diabetes.     

Species variation and plasmid incidence among fluorescent Pseudomonas strains isolated from agricultural and industrial soils

Abstract: A total of 132 different fluorescent Pseudomonas strains were isolated from several agricultural and industrial soils. The bacteria from the two different soil environments were compared for species and biotype variation, antibiotic and heavy metal resistance profiles, ability to degrade polyaromatic hydrocarbons, and plasmid incidence. Irrespective of the soil type, the isolates belonged to Pseudomonas fluorescens biotypes I–VI and Pseudomonas putida biotype B. Except for a streptomycin resistant isolate from one of the industrial soils, all the strains had the same antibiotic resistance profile. However, there was a higher incidence of heavy metal resistance and polyaromatic hydrocarbon degradation phenotypes in the isolates from industrial soils than from the agricultural soils. Only 2 out of 68 strains from agricultural soil were found to carry plasmids, while 28 out of 64 strains from industrial soil had plasmids. A majority of the plasmids (56%) were estimated to be larger than 50 kb, indicating that they could encode transfer functions. However, transferability as indicated by the ability to mobilize an IncQ plasmid (tra⁻, mob⁺), was observed with only one plasmid. None of the plasmid(s) containing isolates hybridized to a ³²P-labelled repP probe suggesting that none of the indigenous plasmids in the soil fluorescent Pseudomonas strains was related to the IncP group of conjugative plasmids commonly associated with resistance and catabolic genes.     

Examining glucose transport in single vascular smooth muscle cells with a fluorescent glucose analog

Changes in vascular smooth muscle glucose transport are thought to contribute to the pathogenesis of vascular disease in conditions such as diabetes, yet no single-cell assay for glucose uptake by VSM exists. Therefore, we examined the uptake of the fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) in isolated pig vascular smooth muscle cells (VSMC) using digital imaging microscopy. Uptake of 2-NBDG by VSMC was inhibited by D-glucose but not by L-glucose, suggesting that 2-NBDG enters VSMC via glucose transporters. Uptake of 2-NBDG was linear in the presence of 10 mM D-glucose (n=6, R2=0.9408) but not in its absence (n=4, R2=0.9993), indicating that 2-NBDG is not metabolized and accumulates within the cells. 2-NBDG fluorescence in VSMC was often non-uniform and appeared to represent binding of 2-NBDG to some cytoplasmic component. The present study demonstrates that 2-NBDG is a useful tool for examining vascular smooth muscle glucose uptake at the single cell level.