Sediment deposit thickness and its effect on critical velocity for incipient motion.

The understanding of how the sediment deposit thickness influences the incipient motion characteristic is still lacking in the literature. Hence, the current study aims to determine the effect of sediment deposition thickness on the critical velocity for incipient motion. An incipient motion experiment was conducted in a rigid boundary rectangular flume of 0.6 m width with varying sediment deposition thickness. Findings from the experiment revealed that the densimetric Froude number has a logarithmic relationship with both the thickness ratios ts/d and ts/y0 (ts: sediment deposit thickness; d: grain size; y0: normal flow depth). Multiple linear regression analysis was performed using the data from the current study to develop a new critical velocity equation by incorporating thickness ratios into the equation. The new equation can be used to predict critical velocity for incipient motion for both loose and rigid boundary conditions. The new critical velocity equation is an attempt toward unifying the equations for both rigid and loose boundary conditions.

An electrochemical lipopolysaccharide sensor based on an immobilized Toll-Like Receptor-4.

Infections affect millions of people each year and yet methods to ascertain their cause can take more than 24h to be effective. This delay between the presentation with symptoms and the ability to make an informed decision about treatment can have adverse consequences, including death in severe cases. Additionally, pathogen identification is a concern for public safety amid the growing threat of bioterrorism. Developing a detection system based on the immune system offers the advantage of broad specificity, while still remaining pertinent to human health. In this work, human Toll-Like Receptor-4 (TLR-4), a protein responsible for detecting lipopolysaccharide (LPS) of Gram-negative bacteria, was immobilized on both a large area and micro gold electrode via the tethering interaction of a modified Self-Assembled Monolayer (mSAM). In response to varying concentrations of its target, the protein-electrode combination showed a logarithmically proportional increased resistance to charge transfer from a solution-based redox probe, due to the formation of TLR-4 protein dimers. It also demonstrated excellent sensitivity to trace levels of Gram-negative bacteria, while remaining insensitive to both Gram-positive and viral challenges. Further characterization of our mSAM revealed that maintaining the appropriate receptor orientation on the electrode surface, mimicking TLR-4's role in a cellular context, was essential in producing a responsive sensor.

Involvement of endothelium/nitric oxide in vasorelaxation induced by purified green tea (-)epicatechin.

The present study investigated the involvement of endothelial nitric oxide in relaxation induced by purified green tea (-)epicatechin in rat isolated mesenteric arteries. (-)Epicatechin caused both endothelium-dependent and -independent relaxation. NG-Nitro-L-arginine methyl ester (L-NAME, 100 microM) and methylene blue (10 microM) significantly attenuated (-)epicatechin-induced relaxation in endothelium-intact tissues. L-Arginine (1 mM) partially antagonized the effect of L-NAME. (-)Epicatechin-induced relaxation was inhibited by Rp-guanosine 3',5'-cyclic monophosphothioate triethylamine. In contrast, indomethacin and glibenclamide had no effect. (-)Epicatechin (100 microM) significantly increased the tissue content of cyclic GMP and NG-nitro-L-arginine (100 microM) or removal of the endothelium abolished this increase. (-)Epicatechin (100 microM) induced an increase in intracellular Ca2+ levels in cultured human umbilical vein endothelial cells. Iberiotoxin at 100 nM attenuated (-)epicatechin-induced relaxation in endothelium-intact arteries and this effect was absent in the presence of 100 microM L-NAME. In summary, (-)epicatechin-induced endothelium-dependent relaxation is primarily mediated by nitric oxide and partially through nitric oxide-dependent activation of iberiotoxin-sensitive K+ channels. In addition, there may be a causal link between increased Ca2+ levels and nitric oxide release in response to (-)epicatechin.

Frontal affinity chromatography for the screening of mixtures.

A protein stationary phase for frontal affinity chromatography was prepared, containing biotinylated beta-galactosidase immobilized to controlled pore glass beads via covalently bonded streptavidin. Single microaffinity columns of approximately 30 pmol of active beta-galactosidase were prepared from this material and characterized with a known ligand by frontal analysis. These columns were used to measure the specific interactions between the bound beta-galactosidase and a library of modified beta-galactopyranosides using electrospray mass spectrometry as the means of detection. The library contained 89 entries, each representing 4 diastereomers for a total of 356 library members. A single entry was analysed revealing differential activity among the 4 isomers. The library was grouped into 10 mixtures of 24-40 members each with each mixture infused under frontal chromatographic conditions. This deconvolution procedure led to the identification of 34 entries containing isomers with K(d) values better than 10 microM. A method based on a displacement principle was implemented as a rapid prescreen which served as the basis for a parallel column high throughput screening assay.

Involvement of endothelium in relaxant action of glibenclamide on the rat mesenteric artery.

The present report describes the complex effect of glibenclamide, an antidiabetic sulfonylurea agent, on the rat isolated mesenteric artery. Although glibenclamide concentration dependently reversed the relaxant effect of pinacidil, an activator of ATP-sensitive K+ channels (the concentration for half-maximum reversal effect was 0.56 microM with endothelium and 0.17 microM without endothelium), in the artery precontracted with phenylephrine (1 microM), it relaxed phenylephrine-induced sustained contraction at higher concentrations (IC50: 4.4+/-1.1 microM with endothelium and 226.1+/-44.2 microM without endothelium). The relaxant effect of glibenclamide was partially inhibited by pretreatment of the artery with either NG-nitro-L-arginine (10-100 microM) or methylene blue (1 microM). Indomethacin (10 microM) had no effect. Moreover, glibenclamide also concentration dependently (3-500 microM) reduced the sustained contraction induced by 60 mM K+ (IC50: 99.5+/-16.1 microM). The relaxation induced by glibenclamide was not affected by various putative K+ channel blockers such as charybdotoxin (100 nM), tetraethylammonium ions (1 mM), apamin (100 nM) and 4-aminopyridine (1 mM). The results indicate an involvement of the endothelium, probably of nitric oxide, in the relaxation induced by glibenclamide in the endothelium-intact rat mesenteric arteries. The inhibitory effect of glibenclamide on the high-K+-induced contraction suggests that glibenclamide may interfere with Ca2+ influx, which in turn affects intracellular Ca2+ levels in arterial smooth muscle, leading to reduction of muscle contractility. It is suggested that two distinct pharmacological effects induced by glibenclamide may be mediated through different glibenclamide binding sites, however, the data show an overlap of concentrations of glibenclamide for producing the two effects in rat isolated mesenteric arteries.

Role of endothelium in thapsigargin-induced arterial responses in rat aorta.

We assessed the role of endothelium in the arterial response to thapsigargin, the Ca(2+)-ATPase inhibitor of the endoplasmic reticulum, in rat isolated aortic rings. Thapsigargin induced an endothelium-dependent relaxation of phenylephrine-contracted aortic rings with an EC(50) of 2.6+/-0.4 nM and a 75% maximum relaxation, while it was less effective against 30 mM K(+)-induced contraction. Pretreatment of aortic rings with N(G)-nitro-L-arginine methyl ester (30 microM) or methylene blue (1 microM) reduced thapsigargin-induced relaxation by approximately 85%. Thapsigargin failed to relax the endothelium-denuded rings. L-Arginine (3 mM) partially, but significantly, antagonized the effect of 30 microM N(G)-nitro-L-arginine methyl ester. Pretreatment with indomethacin (3 microM), glibenclamide (1 microM) or iberiotoxin (100 nM) did not alter the thapsigargin-induced relaxation. In contrast, pretreatment with tetrapentylammonium ions (TPA(+), 1-3 microM) or with 300 microM Ba(2+) suppressed the relaxant response to thapsigargin. TPA(+) (3 microM) also attenuated acetylcholine-induced relaxation. Thapsigargin-induced endothelium-dependent relaxation was primarily dependent on the presence of extracellular Ca(2+). Interestingly, when the tissues were exposed to very low concentrations of thapsigargin (1-3 nM) the nitric oxide-dependent relaxation induced by acetylcholine or A23187 was markedly reduced. While thapsigargin (3 nM) did not influence the relaxation induced by endothelium-independent dilators, sodium nitroprusside and verapamil. These results indicate that thapsigargin produced complex vascular effects primarily by acting on the endothelial cells. Thapsigargin causes an endothelial nitric oxide-dependent relaxation; on the other hand, it inhibits nitric oxide-mediated relaxation at the similar concentrations. Activation of TPA(+)- and Ba(2+)-sensitive but not Ca(2+)-activated or ATP-sensitive K(+) channels may be also involved in thapsigargin-induced relaxation of rat isolated aortic rings.

Abolition of endothelium-dependent relaxation in the rat aorta by tetraoctylammonium ions.

Quaternary ammonium ions are common pharmacological probes used to study the kinetic properties of K+ channels in smooth muscle cells. On the other hand, some ammonium compounds cause vasorelaxation through unknown mechanisms. The main aim of this study was to examine a unique role of endothelium in the vascular response to tetraoctylammonium ions (TOA+) in the isolated rat aorta. Changes in contractile force were measured by force transducers and total tissue content of cGMP was measured by radioimmunoassay. Endothelial cytosolic Ca2+ ([Ca2+]i) was assessed by laser scanning confocal microscopy.

Prejunctionally mediated inhibition of neurotransmission by isoprenaline in rat vas deferens.

Effects of isoprenaline on monophasic contractions evoked by electric field stimulation were studied in rat isolated prostatic vas deferens. Isoprenaline reduced electrically evoked contractions (EC50: 0.27 +/- 0.05 microM), and propranolol concentration-dependently antagonized the effect of isoprenaline. In contrast, isoprenaline (0.3-3 microM) did not affect the contractile response induced by exogenous noradrenaline or ATP, while forskolin (100 nM) attenuated agonist-induced contraction. In some tissues, adrenergic and purinergic components of the electrically evoked contraction were isolated by exposure to alpha,beta-methylene ATP (3 microM) and prazosin (3 microM), respectively. Isoprenaline induced a greater inhibition of purinergic than adrenergic component of the electrically evoked contraction. Iberiotoxin (50 nM), glibenclamide (3 microM), 4-aminopyridine (0.3 mM) and tetraethylammonium ions (1 mM) attenuated the effect of isoprenaline. These results indicate that isoprenaline-induced inhibition of the electrically evoked (both purinergic and adrenergic) contraction was mediated primarily through activation of prejunctional beta-adrenoceptors, which probably inhibited release of contractile transmitters from sympathetic nerves supplying vas deferens. Lack of effect of isoprenaline on agonist-induced contraction does not favour a functional role of beta-adrenoceptors in vas smooth muscle.

Co-composting of soybean residues and leaves in Hong Kong.

The goal of this project was to evaluate the feasibility of co-composting of soybean residues and leaves and the effects of turning frequency on compost quality. Soybean residues were mixed with leaves and sawdust in 1:1:3 (w/w wet weight) for achieving a C/N ratio of about 30. Three heaps of about 4 m3 of compost mixtures were prepared receiving a turning frequency of daily (pile A), 3-day (pile B) and weekly (pile C) turning. Different turning frequencies did not significantly affect the changes in pH and volatile solids throughout the composting period. High turning frequency caused a lower electrical conductivity and NH4-N contents as well as a shorter duration of thermophilic phase, because of a high heat loss by evaporation and volatilization of ammonia in the pile. The highest C decomposition of 4% occurred in the pile with a 3-day turning period, which coincided with the higher-nitrogen content in this treatment. All treatments with different turning frequencies reached maturation at 63 days as indicated by the soluble organic carbon, soluble NH4-N, C/N ratio and cress seed germination index. However, increasing the aeration during composting period was beneficial in accelerating the maturation process. Taking into consideration less labour and lower operation costs as compared to daily turning, it can be suggested that a 3-day turning frequency would be more appropriate for reaching acceptable quality of compost and ease in operation.

Choice and constraints in floodplain occupation: the influence of structural factors on residential location in peninsular Malaysia.

In Peninsular Malaysia 'structural' factors are found to influence strongly people's persistent occupation of floodplains. Thus, despite a high level of flood hazard awareness, a high level of pessimism and a high level of expectation of future floods, poorer individuals seldom attempt to leave for more advantageous locations but are instead trapped in their present locations by structural factors such as poverty, low residential and occupational mobility, low educational attainment, traditional land inheritance, government aid, and government disaster preparedness, relief and rehabilitation programmes. These forces exert a strong influence upon individuals and largely control their choice of residential location in response to flood hazards, thereby reinforcing the persistent occupation of floodplains. Structural factors such as landlessness, rural-urban migration, floodplain encroachment and squatting are also highly influential in leading people to move. Even for those who move, structural factors have largely confined their choice of residential location to urban floodplains.

Institutional arrangements for flood hazard management in Malaysia: an evaluation using the criteria approach.

Institutional aspects of flood hazards significantly affect their outcomes in Malaysia. Institutional arrangements to deal with floods include: legislative activity, organisational structures, attitudes and sub-culture, and policies and instruments. When assessed in terms of four specific criteria, institutional aspects of flood hazards are found to be largely inadequate. Disaster reduction programmes are over-dependent on a reactive approach based largely on technology and not even aimed at floods specifically. Structural flood reduction measures are the predominant management tool and, although the importance of non-structural measures is recognised, thus far they have been under-employed. Current laws and regulations with regard to flood management are also insufficient and both the financial and human resources of flood hazard organisations are generally found to be wanting. Finally, economic efficiency, equity and public accountability issues are not adequately addressed by institutional arrangements for flood hazards.

Intracellular inhibition of blood group A glycosyltransferase.

We report the intracellular inhibition of blood group A N-acetylgalactosaminyltransferase in the human colorectal carcinoma cell line HT29 by 3-amino-3-deoxy-[Fucalpha(1-2)]Galbeta-O(CH2)7CH3. Inhibition was demonstrated with a novel capillary electrophoresis assay that monitored decreased intracellular conversion of fluorescently labelled Fucalpha(1-2)Gal-R acceptor to the corresponding A epitope, GalNAcalpha(1-3)[Fucalpha(1-2)]Galbeta-R. Growth of HT29 cells with either the amino-inhibitor or a competitive substrate, Fucalpha(1-2)Galbeta-O(CH2)7CH3, also resulted in decreased expression of blood group A determinants on cell-associated glycoproteins, as detected by immunoprecipitation analysis using A-specific monoclonal antibodies. Furthermore, exposure of these cells to the amino-inhibitor or competitive substrate resulted in significant reduction of cell-surface expression of blood group A determinants. As integrin alpha3beta1, a cell-surface receptor mediating cell-cell and cell-extracellular matrix interactions, was shown previously to be a major carrier of blood group A determinants on HT29 cells, the studies described herein highlight the potential usefulness of these compounds for elucidating the role of blood group A determinants in biological phenomena.

Contractile and relaxant effects of tetrapentylammonium ions in rat isolated mesenteric artery.

Both contractile and relaxant responses to tetrapentylammonium ions (TPA+) were studied in rat isolated mesenteric artery. TPA+ (5-10 micromol/l) caused a sustained increase of muscle tension. The contractile effect of TPA+ (10 micromol/l) was dependent upon the presence of extracellular Ca2+ but independent of the presence of endothelium. TPA+ (10-50 micromol/l) induced biphasic contraction, and the amplitude of peak and sustained tension decreased with increasing TPA+ concentration. TPA+ (100-300 micromol/l) only produced monophasic contraction. TPA+ (50 micromol/l) abolished the transient contraction induced by caffeine (10 mmol/l) or phenylephrine (1 micromol/l) in the absence of extracellular Ca2+. Nifedipine and verapamil concentration-dependently reduced the TPA+-induced contraction with respective IC50 values of 1.34 +/- 0. 24 and 9.46 +/- 1.36 nmol/l, these values were similar to 1.35 +/- 0. 21 and 16.07 +/- 1.71 nmol/l, respectively, for the inhibitory effects of nifedipine and verapamil on the high K+ (60 mmol/l)-induced contraction. TPA+ (>10 micromol/l) concentration-dependently reduced the phenylephrine (1 micromol/l)-, U46619 (30 nmol/l)-, endothelin I (10 nmol/l)- and high K+ (60 mmol/l)-induced sustained tension with respective IC50 values of 53. 7 +/- 9.5, 31.9 +/- 5.3, 30.9 +/- 3.4 and 20.9 +/- 2.8 micromol/l. The present results indicate that TPA+ at low concentrations could contract the arterial smooth muscle probably through promoting Ca2+ influx. At higher concentrations (>20 micromol/l), TPA+ relaxes arterial smooth muscle probably through inhibition of both nifedipine-sensitive Ca+ channels and internal Ca2+ release. TPA+, unlike other quaternary ammonium ions, could therefore act at multiple sites in arterial smooth muscle.

A protein kinase G-sensitive channel mediates flow-induced Ca(2+) entry into vascular endothelial cells.

The hemodynamic force generated by blood flow is considered to be the physiologically most important stimulus for the release of nitric oxide (NO) and prostacyclin (PGI(2)) from vascular endothelial cells (1). NO and PGI(2) then act on the underlying smooth muscle cells, causing vasodilation and thus lowering blood pressure (2, 3). One critical early event occurring in this flow-induced regulation of vascular tone is that blood flow induces Ca(2+) entry into vascular endothelial cells, which in turn leads to the formation of NO (4, 5). Here we report a mechanosensitive Ca(2+)-permeable channel in vascular endothelial cells. The activity of the channel was inhibited by 8-Br-cGMP, a membrane-permeant activator of protein kinase G (PKG), in cell-attached membrane patches. The inhibition could be reversed by PKG inhibitor KT5823 or H-8. A direct application of active PKG in inside-out patches blocked the channel activity. Gd(3+), Ni(2+), or SK&F-96365 also inhibited the channel activity. A study of fluorescent Ca(2+) entry revealed a striking pharmacological similarity between the Ca(2+) entry elicited by flow and the mechanosensitive Ca(2+)-permeable channel we identified, suggesting that this channel is the primary pathway mediating flow-induced Ca(2+) entry into vascular endothelial cells.

Cloning and heterologous expression of an alpha1,3-fucosyltransferase gene from the gastric pathogen Helicobacter pylori.

Helicobacter pylori is an important human pathogen which causes both gastric and duodenal ulcers and is also associated with gastric cancer and lymphoma. This microorganism has been shown to express cell surface glycoconjugates including Lewis X (Lex) and Lewis Y. These bacterial oligosaccharides are structurally similar to tumor-associated carbohydrate antigens found in mammals. In this study, we report the cloning of a novel alpha1,3-fucosyltransferase gene (HpfucT) involved in the biosynthesis of Lex within H. pylori. The deduced amino acid sequence of HpfucT consists of 478 residues with the calculated molecular mass of 56,194 daltons, which is approximately 100 amino acids longer than known mammalian alpha1,3/1,4-fucosyltransferases. The approximately 52-kDa protein encoded by HpfucT was expressed in Escherichia coli CSRDE3 cells and gave rise to alpha1,3-fucosyltransferase activity but neither alpha1,4-fucosyltransferase nor alpha1,2-fucosyltransferase activity as characterized by radiochemical assays and capillary zone electrophoresis. Truncation of the C-terminal 100 amino acids of HpFuc-T abolished the enzyme activity. An approximately 72-amino acid region of HpFuc-T exhibits significant sequence identity (40-45%) with the highly conserved C-terminal catalytic domain among known mammalian and chicken alpha1,3-fucosyltransferases. These lines of evidence indicate that the HpFuc-T represents the bacterial alpha1,3-fucosyltransferase. In addition, several structural features unique to HpFuc-T, including 10 direct repeats of seven amino acids and the lack of the transmembrane segment typical for known eukaryotic alpha1,3-fucosyltransferases, were revealed. Notably, the repeat region contains a leucine zipper motif previously demonstrated to be responsible for dimerization of various basic region-leucine zipper proteins, suggesting that the HpFuc-T protein could form dimers.

Role of endothelium and K+ channels in dobutamine-induced relaxation in rat mesenteric artery.

1. In order to examine the possible involvement of the endothelium and K+ channel activation in the relaxation induced by dobutamine, a beta 1-adrenoceptor agonist, in rat isolated mesenteric arteries, the effects of inhibitors of nitric oxide (NO) activity, blockers of K+ channels and high extracellular K+ were studied by measuring isometric tension in both endothelium-intact and -denuded arteries. 2. Dobutamine inhibited the phenylephrine (PE)-induced sustained tension with a pEC50 of 7.40 +/- 0.08 in endothelium-intact arteries. Removal of functional endothelium attenuated the effect of dobutamine. The relaxation induced by dobutamine was inhibited by the beta 1-adrenoceptor antagonist CGP 20712A (3 mumol/L) but not by the beta 2-adrenoceptor antagonist ICI 118,551 (3 mumol/L) in endothelium-denuded arteries. 3. Pretreatment with NG-nitro-L-arginine (L-NNA; 100 mumol/L) or methylene blue (3 mumol/L) induced a similar degree of inhibition of the dobutamine-induced relaxation in endothelium-intact arteries, while NG-nitro-D-arginine (100 mumol/L) and indomethacin (10 mumol/L) had no effect. In contrast, pretreatment with L-NNA (100 mumol/L) did not affect the relaxation induced by sodium nitroprusside (SNP) or forskolin. Methylene blue (3 mumol/L) inhibited the relaxant response to SNP. 4. Charybdotoxin (CTX; 100 nmol/L), iberiotoxin (IBX; 100 nmol/L) and tetraethylammonium ions (TEA+; 3 mmol/L) significantly reduced the dobutamine-induced relaxation. Tetrapentylammonium ions (TPA+; 5 mumol/L) markedly inhibited the relaxant effect of dobutamine. The pEC50 values for control and in the presence of TPA+ in endothelium-intact arteries were 7.35 +/- 0.11 and 6.14 +/- 0.17, respectively, and 6.35 +/- 0.09 and 5.87 +/- 0.17 for control and in the presence of TPA+ in endothelium-denuded arteries, respectively. In contrast, glibenclamide (3 mumol/L) was ineffective. At 5 mumol/L, TPA+ also inhibited the relaxation induced by forskolin. 5. The maximal relaxation of PE-contracted arteries induced by 3 mumol/L dobutamine was completely abolished in the 60 mmol/L K(+)-contracted arteries with and without endothelium, while dobutamine at a concentration greater than 3 mumol/L induced inhibition of the high-K+ response. 6. The present results indicate that endothelium, probably NO but not prostacyclin, was involved in the dobutamine-induced relaxation in rat mesenteric arteries. Activation of CTX-, IBX- and TPA(+)-sensitive K+ channels contributed towards the observed relaxation. Loss of the ability to relax the 60 mmol/L K(+)-contracted arteries suggests that endothelium-derived vasoactive factors affected by concentrations of dobutamine less than 3 mumol/L may also act through K+ channels in our preparations. Higher concentrations of dobutamine may have a direct, endothelium-independent relaxant effect.

A novel viral alpha2,3-sialyltransferase (v-ST3Gal I): transfer of sialic acid to fucosylated acceptors.

The substrate specificity of an alpha2,3-sialyltransferase (v-ST3Gal I) obtained from myxoma virus infected RK13 cells has been determined. Like mammalian sialyltransferase enzymes, the viral enzyme contains the characteristic L- and S-sialyl motif sequences in its catalytic domain. Analysis of the deduced amino acid sequences of cloned sialyltransferases suggests that v-ST3Gal I is closely related to mammalian ST3Gal IV. v-ST3Gal I catalyzes the transfer of sialic acid from CMP-NeuAc to Type I (Galbeta1-3GlcNAcbeta) II (Galbeta1-4GlcNAcbeta) and III (Galbeta1-3GalNAcbeta) acceptors. In addition, the viral enzyme also transfers sialic acid to the fucosylated acceptors Lewis(x) and Lewis(a). This substrate specificity is unlike any sialyltransferases described to date, though it is most comparable with those of mammalian ST3Gal IV enzymes. The products from reactions with fucosylated acceptors were characterized by capillary zone electrophoresis, (1)H-NMR spectroscopy and mass spectrometry. They were shown to be 2,3-sialylated Lewis(x) and 2,3-sialylated Lewis(a), respectively.

Single-cell analysis avoids sample processing bias.

Microscale separation tools such as capillary chromatography and capillary electrophoresis (CE) allow the study of metabolism in individual cells. In this work, we demonstrate that single-cell analysis describes metabolism more accurately than analysis of cellular extracts. We incubated HT29 cells (human colon adenocarcinoma) with a fluorescently labeled metabolic probe. This disaccharide, LacNAc, was labeled with a fluorescent dye, tetramethylrhodamine (TMR). The probe was taken up by the cells and metabolized to a number of products that retained the fluorescent label. We then split the cells into two batches. A cellular extract was prepared from one batch and analyzed by CE with laser-induced fluorescence (LIF) detection. The cells from the second batch were used for single-cell analysis by CE-LIF. Separation and detection conditions were identical for extract and single-cell analyses. We found that the electropherogram obtained by averaging the results from a number of single cells differed significantly from the cell extract electropherogram. Differences were due to sample processing during extract preparation. Disruption of the cells liberated enzymes that were compartmentalized within the cell, which allowed non-metabolic reactions to proceed. The accumulation of these non-metabolic products introduced a bias in the cell extract assay. During single-cell analysis, cells were lysed inside the capillary and the separation voltage was applied immediately to separate the enzymes from their substrates and prevent non-metabolic reactions. This paper is the first to report that CE analysis of single cells provides more accurate metabolic information than the CE analysis of a cellular extract.

The biosynthesis of Lewis X in Helicobacter pylori.

The biosynthesis of the Lewis X determinant (Gal beta 1-4 [Fuc alpha 1-3]GlcNAc beta-) in three strains of Helicobacter pylori has been investigated. Strains UA 861, UA 802 and UA 1182 contain alpha 1,3 fucosyltransferase and beta 1,4 galactosyltransferase activities that synthesize the Lewis X structure by the transfer of monosaccharides from GDP-fucose and UDP-galactose donors, respectively. The enzyme reaction products that formed were characterized by capillary zone electrophoresis and by 1H-NMR spectroscopy. The biosynthetic pathway is therefore identical to that found in humans. In the three strains, the fucosyltransferase and galactosyltransferase activities differed in various cellular fractions. Km values for their donor and acceptor substrates also differed.

Correlating cell cycle with metabolism in single cells: combination of image and metabolic cytometry.

BACKGROUND: We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. METHODS: A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. RESULTS: Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells. CONCLUSIONS: This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.