Comparative studies of glucose-fed and glucose-starved hamster cell cultures: responses in galactose metabolism.

The metabolic flow of trace amounts of D-[14C]-galactose was followed in cultures of transformed and untransformed hamster cells over a period ranging from five minutes to two hours. The results of chromatographic and enzymatic analyses of the soluble pools are described. Non-glycolytic cells(previously deprived of sugar periods of up to 24 hours) convert D-galactose to galactose-1-phosphate and uridine diphosphoglucuronic acid in 10 to 20 minutes. In the same short assay time, glycolytic cells which have been maintained for 24 hours in media containing glucose or galactose convert D-galactose to uridine diphsphogalactose and uridine diphosphoglucose (ratio 1.4:1). Long term diprivation of sugar also results in 3- to 4-fold increases in the uptake of galactose. In addition, the incorporation of galactose label into chloroformethanol soluble material appears to be influenced by the culture conditions of the untransformed cells while incorporation in the transformed cells appears unaffected. When cycloheximide is included in the maintenance medium for extended periods, the non-glycolytic cells also show increases in galactose uptake rates but the glucose-fed, glycolytic cells llose uptake ability. UDPhexose is the main galactose metabolic peak in the soluble pools of the cycloheximide-treated, glycolytic and the cycloheximide-treated, non-glycolytic cells. The results of these experiments suggests that uptake of galactose and its subsequent metabolism are under separate control.     

Carbohydrate utilization patterns and substrate preferences in Bacteroides thetaiotaomicron

Specific growth rates of Bacteroides thetaiotaomicron NCTC 10582 with either glucose, arabinose, mannose, galactose or xylose as sole carbon sources were 0.42/h, 0.10/h, 0.38/h, 0.38/h and 0.16/h respectively, suggesting that hexose metabolism was energetically more efficient than pentose fermentation in this bacterium. Batch culture experiments to determine whether carbohydrate utilization was controlled by substrate-induced regulatory mechanisms demonstrated that mannose inhibited uptake of glucose, galactose and arabinose, but had less effect on xylose. Arabinose and xylose were preferentially utilized at high dilution rates (D > 0.26/h) in carbon-limited continuous cultures grown on mixtures of arabinose, xylose, galactose and glucose. When mannose was also present, xylose was co-assimilated at all dilution rates. Under nitrogen-limited conditions, however, mannose repressed uptake of all sugars, showing that its effect on xylose utilization was strongly concentration dependent. Studies with individual D-ZU-14C]-labelled substrates showed that transport systems for glucose, galactose, xylose and mannose were inducible. Measurements to determine incorporation of these sugars into trichloroacetic acid-precipitable material indicated that glucose and mannose were the principal precursor monosaccharides. Xylose was only incorporated into intracellular macromolecules when it served as growth substrate. Phosphoenolpyruvate:phosphotransferase systems were not detected in preliminary experiments to elucidate the mechanisms of sugar uptake, and studies with inhibitors of carbohydrate transport showed no consistent pattern of inhibition with glucose, galactose, xylose and mannose. These results indicate the existence of a variety of different systems involved in sugar transport in B. thetaiotaomicron.     

Effects of 2-deoxy-D-glucose on the synthesis of RNA and protein in Saccharomyces carlsbergensis G-517

When Saccharomyces carlsbergensis G-517 was grown in 10 mM galactose as the carbon source, the addition of 2-deoxy-D-glucose restricted the uptake of galactose, [3H]uridine and [3H]leucine, and restricted invertase synthesis (beta-D-fructofuranoside fructohydrolase; EC 3.2.1.26) for a period of 60-90 min. During this time, the radioactive antimetabolite was taken up by the cells; afterwards, invertase synthesis was enhanced, and the utilizaton rate of galactose, [3H]uridine and [3H]leucine increased until it reached that of the control culture. When glucose was used as a carbon source, sugar utilization and uptake of radioactive precursors were unaffected by addition of the deoxysugar.     

Selective high metabolic lability of uridine, guanosine and cytosine triphosphates in response to glucose deprivation and refeeding of untransformed and polyoma virus-transformed hamster fibroblasts.

Sugar deprivation of hamster fibroblasts (NIL) affected the steady state levels (pool sizes) of cellular acid soluble nucleotides in the folloing fashion: the pools of UTP, GTP and CTP decreased to a much greater extent than the cellular ATP pools, with the UTP pools undergoing the most dramatic reduction. Sugar deprivation of polyoma-transformed NIL cells (PyNIL) yielded even sharper decreases in the nucleoside triphosphate pools with relative changes similar to those of the untransformed cells. Inhibition of protein synthesis by cycloheximide, initiated at the onset of (and continued during) sugar deprivation, prevented the reduction in pool sizes and yielded values slightly higher than those observed for pool sizes in cells cultured in sugar-supplemented medium.Refeeding glucose to sugar-depleted hamster fibroblasts led to rapid increases (within 1 hour) in the UTP and CTP pools to levels well above the pool sizes observed in cells which were continuously cultured (16 hours) in sugar supplemented medium. Feeding NIL or PyNIL cells with fructose instead of glucose as the only hexose source did not appreciably affect any of the ribonucleoside triphosphate pool sizes. Measurements of hexose uptake by NIL and PyNIL cells under a variety of conditions suggest that hexose transport is not regulated by the total cellular pools of ATP or any of the other ribonucleoside triphosphates.     

Analysis of kinetic uptake phenomena of monosaccharide and disaccharide by suspension TBY-2 cells using an FT-IR/ATR method

The influence of sugars in culture media on the kinetics of the mono- and disaccharide uptake and cell growth behavior was studied by mid-infrared spectroscopy using a Fourier transform infrared spectrometer (FT-IR) equipped with an attenuate total reflection accessory (ATR). We performed the plant cell cultivation with Nicotiana tabacum cv. Bright Yellow No.2 (TBY-2) cells in the culture media, which contained glucose, fructose, mannose, galactose, sucrose, trehalose, maltose or lactose. Consequently, the differences of the kinetic sugar uptake and cell growth behavior among all the cultivations were confirmed. In particular, a very long lag time before the galactose uptake was observed, and the spectral-pattern of the maltose medium presented almost the same as the initial one during the cultivation. Furthermore, base on the non-dimensional cultivation time for cell growth behavior, it was suggested that the TBY-2 cells consumed sugar before cell growth and produced the ethanol just after cell growth.     

Catabolite inhibition and sequential metabolism of sugars by Streptococcus lactis.

Growth of galactose-adapted cells of Streptococcus lactis ML(3) in a medium containing a mixture of glucose, galactose, and lactose was characterized initially by the simultaneous metabolism of glucose and lactose. Galactose was not significantly utilized until the latter sugars had been exhausted from the medium. The addition of glucose or lactose to a culture of S. lactis ML(3) growing exponentially on galactose caused immediate inhibition of galactose utilization and an increase in growth rate, concomitant with the preferential metabolism of the added sugar. Under nongrowing conditions, cells of S. lactis ML(3) grown previously on galactose metabolized the three separate sugars equally rapidly. However, cells suspended in buffer containing a mixture of glucose plus galactose or lactose plus galactose again consumed glucose or lactose preferentially. The rate of galactose metabolism was reduced by approximately 95% in the presence of the inhibitory sugar, but the maximum rate of metabolism was resumed upon exhaustion of glucose or lactose from the system. When presented with a mixture of glucose and lactose, the resting cells metabolized both sugars simultaneously. Lactose, glucose, and a non-metabolizable glucose analog (2-deoxy-d-glucose) prevented the phosphoenolpyruvate-dependent uptake of thiomethyl-beta-d-galactopyranoside (TMG), but the accumulation of TMG, like galactose metabolism, commenced immediately upon exhaustion of the metabolizable sugars from the medium. Growth of galactose-adapted cells of the lactose-defective variant S. lactis 7962 in the triple-sugar medium was characterized by the sequential metabolism of glucose, galactose, and lactose. Growth of S. lactis ML(3) and 7962 in the triple-sugar medium occurred without apparent diauxie, and for each strain the patterns of sequential sugar metabolism under growing and nongrowing conditions were identical. Fine control of the activities of preexisting enzyme systems by catabolite inhibition may afford a satisfactory explanation for the observed sequential utilization of sugars by these two organisms.     

Respiration-Dependent Utilization of Sugars in Yeasts: a Determinant Role for Sugar Transporters

In many yeast species, including Kluyveromyces lactis, growth on certain sugars (such as galactose, raffinose, and maltose) occurs only under respiratory conditions. If respiration is blocked by inhibitors, mutation, or anaerobiosis, growth does not take place. This apparent dependence on respiration for the utilization of certain sugars has often been suspected to be associated with the mechanism of the sugar uptake step. We hypothesized that in many yeast species, the permease activities for these sugars are not sufficient to ensure the high substrate flow that is necessary for fermentative growth. By introducing additional sugar permease genes, we have obtained K. lactis strains that were capable of growing on galactose and raffinose in the absence of respiration. High dosages of both the permease and maltase genes were indeed necessary for K. lactis cells to grow on maltose in the absence of respiration. These results strongly suggest that the sugar uptake step is the major bottleneck in the fermentative assimilation of certain sugars in K. lactis and probably in many other yeasts.     

Hexose transport regulation in cultured hamster cells

Hamster (nil) cells maintained overnight in culture medium containing cycloheximide and either glucose or fructose exhibit strikingly different rates of hexose transport and metabolism (i.e., uptake). Pretreatment of cultures with sulfhydryl reagents makes it possible to determine initial transport rates for a physiological sugar such as galactose which is a catabolite in hamster cells. Using galactose transport as a model, hexose uptake enhancements can now be shown to be due almost entirely to increase in the rate of the transport step. The transport regulation can best be accounted for by a model comprised of 2 antagonizing mechanisms. This model involves turnover of transport carriers as well as inhibitory units (“regulators”). The experimental as well as the theoretical model may also apply to the well-known uptake enhancements observed in oncogenically transformed cells.     

The Carbohydrate Nutrition of Tomato Roots: VI. The Inhibition of Excised Root Growth by Galactose and Mannose and its Reversal by Dextrose and Xylose

The addition of 0.01–0.015 per cent. galactose or 0.005–0.01per cent, mannose reduces by 50 per cent, the linear growthof excised tomato roots cultured in a I per cent, sucrose medium.An addition of 0.03–0.04 per cent, of either sugar causesnot less than a 90 per cent, inhibition of growth. The survivalof meriste-matic activity is higher in presence of fully inhibitoryconcentrations of mannose than of galactose. Roots inhibitedby galactose are distinguishable from those inhibited by mannose. The inhibitory effect of concentrations of galactose up to 0.15per cent, and of mannose up to 0.4 per cent, can be fully antagonizedby the simultaneous addition to the culture medium of dextrose.The minimum ratio of dextrose: inhibitory sugar for maximumantagonism of the growth inhibition is with galactose 5: 1 andwith mannose 3.5: 1. Growth of roots in a dextrose-containingmedium does not protect them from subsequent inhibition by eithergalactose or mannose. d-xylose has significant activity as an antagonist of mannoseinhibition and even more so of galactose inhibition. However,the restoration of growth achievable from the addition of xyloseis not comparable with that resulting from the addition of dextrose.The inhibition of growth by xylose is not alleviated by thesimultaneous addition of dextrose. Maltose has low activityas an antagonist of galactose and mannose inhibition. All othersugars tested and the sugar alcohols corresponding to galactoseand mannose were quite inactive as antagonists of the growthinhibition by these two sugars. Mixtures of partially inhibitory concentrations of galactoseand mannose were less inhibitory than their more inhibitorycomponent. The concentration of dextrose required to reversethe inhibitory activity of such mixtures was not greater thanthe minimum concentration required to antagonize the actionof the more inhibitory component. The antagonism of galactose inhibition by dextrose is not dueto dextrose impeding galactose uptake.     

Effect of acute and chronic vanadate administration on sugar transport in rat jejunum

Vanadate is known to have an insulin-like action which stimulates sugar transport in some systems like adipocytes and muscle cells, but in other systems it inhibits sugar transport by decreasing the activity of (Na+ +K+)-ATPase. To evaluate whether these two opposing actions may influence sugar transport across the intestine, we studied the effects of acute and chronic vanadate administration on the uptake of glucose, galactose, and 3-O-methylglucose in isolated rat intestinal cells. The sugar uptake measurements were also coupled by determinations of rubidium-86 uptake as a measure of the activity of the Na-K pump. Both acute and chronic vanadate administration reduced rubidium uptake by the cells but the reduction did not uniformly influence the uptake of the three sugars in question which were stimulated by the acute exposure of the cells to vanadate. Glucose uptake was also stimulated by chronic vanadate administration, but the uptakes of galactose and 3-O-methylglucose were respectively unaffected or inhibited by chronic vanadate. The findings suggest that the effect of vanadate on sugar transport is dependent on the net difference between two actions of vanadate: (i) stimulation of a receptor site (possibly an insulin receptor site) in the intestinal cell membrane and (ii) inhibition of the Na-K pump. During acute vanadate exposure, the stimulation of the receptor site was very likely a dominant feature which overwhelms the inhibition of the pump. Chronic exposure to vanadate led, on the other hand, to only a limited degree of stimulation of the receptor site and the inhibition of the Na-K pump became evident in the uptake measurements of galactose and 3-O-methyl-glucose. Glucose uptake, however, was stimulated by chronic vanadate ingestion due, very likely, to an increase in the metabolism of this sugar which occurred only with prolonged exposure of the rat intestine to vanadate.     

Galactose Transport in Saccharomyces cerevisiae III. Characteristics of Galactose Uptake in Transferaseless Cells: Evidence Against Transport-Associated Phosphorylation

The characteristics of the inducible galactose transport system in bakers' yeast were studied in uridine diphosphate, galactose-1-phosphate uridylyl-transferaseless cells. Transferaseless cells transport galactose at the same initial rate as wild-type cells and accumulate a mixture of free galactose and galactose-1-phosphate. The addition of ¹⁴C-labeled galactose to cells preloaded with unlabeled galactose and galactose-1-phosphate results in a higher rate of labeling of the free-sugar pool than of the galactose-1-phosphate pool. These results support other evidence that galactose uptake in bakers' yeast is a carrier-mediated, facilitated diffusion and that phosphorylation is an intracellular event after uptake of the free sugar.     

Sugar uptake analysis of suspension Arabidopsis, tobacco, and rice cells in various media using an FT-IR/ATR method

The kinetic behavior of the sugar uptake phenomena of a suspension of Arabidopsis cells was investigated by mid-infrared spectroscopy using Fourier transform infrared spectrometers and attenuated total reflection techniques. The kinetic behavior of the cell growth was also studied and the growth and the sugar uptake behaviors were discussed for three typical plant cells (Arabidopsis, TBY-2, and rice cells). The cell growth rate and the lag period were influenced by not only the types of the plant cells, but also the sugar species used as the carbon source. The characteristics of the sugar uptake behavior were clarified based on the difference in the three types of plant cells. The cell growth and the sugar uptake progressed at approximately the same time in the TBY-2 cells. In the rice cells, the sugar uptake rate was relatively lower than that of the others. On the other hand, the sugar uptake of the Arabidopsis cells started before the cell growth. Furthermore, glucose as the carbon source of the Arabidopsis cell cultivation seems to significantly influence the sugar metabolism. Glucose had a significant influence on the sugar metabolism of the other sugar under the conditions for the mixture of glucose and the other sugar. The characteristics of the sugar uptake phenomena based on the cell growth stage was typical for each plant cell except for some sugars, such as galactose and trehalose, and the behavior of the total sugar uptake had not changed. These results suggested that the cell growth and the sugar uptake in the plant cell cultivation processes may be controlled by the combined supply of the sugar species as the carbon source. The detailed data for plant cell cultivation using each sugar obtained in this study would be useful for bioscience research and for cultivation process control using various sugars, for example, purified or sugar mixtures formed from biomass materials.     

Effect of anaerobiosis, dinitrophenol and fluoride on the active intestinal transport of galactose in snail.

The active transport of galactose across the intestinal wall (everted sacs) of the snail Cryptomphalus hortensis Müller has been studied in vitro, under several metabolic conditions. Anaerobiosis does not change the serosal/mucosal galactose gradients which are developed in oxygen atmosphere. Dinitrophenol (10(-4) M) greatly increased the O2 uptake by the tissue and clearly inhibits the sugar transport. At 5 times 10(-4) M concentration, DNP totally prevents the uphill transport while the O2 uptake is normal. The inhibition produced by DNP does not increase by anaerobiosis. Fluoride inhibits the galactose transport and also the O2 uptake. It is deduced that in snail intestine the energy for the active transport of galactose can be supplied by aerobic as much as by anaerobic metabolism. The inhibition by dinitrophenol seems to be independent of its uncoupling action on the oxidative phosphorylation. The inhibitory effect of NaF may be due both to glycolisis inhibition and to alteration of the digestive epithelium.     

Transport and phosphorylation of D-galactose in renal cortical cells.

An improved analytical procedure for the extraction and determination of total, free and phosphorylated tissue sugar is described. This method, employing ZnSO4 plus Ba(OH)2 for the precipitation of sugar phosphates, yields values identical with those obtained by the more laborious separation of free and phosphorylated sugar by ion-exchange chromatography. Erroneous values for free sugar due to the action of a Zn2+ -activated phosphatase and/or the lability to acids of some sugar phosphates, are avoided. Using this technique for the sudy of transport and phosphorylation of D-galactose in rabbit renal cortical slices and tissue extracts, it was found: 1. The cellular uptake of D-galactose was associated with the appearance of both free and phosphorylated sugar whether or not external Na+ was present. At 1 mM sugar, galactose was accumulated in the cells against a modest concentration gradient of 1.445 +/- 0.097 (n = 17). Galactose phosphate appeared in the cells considerably faster than free sugar under conditions of net uptake as well as of steady-state exchange (pulse-labelling). 2. Increasing saline pH (6-8) increased the cellular levels of sugar phosphate without affecting the steady-state values of free sugar. With tissue extracts, increasing pH also stimulated the activity of galactokinase and the dephosphorylation of galactose 1-phosphate by a Zn2+ -activated phosphatase. 3. 0.5 mM phlorizin inhibited the tissue uptake of galactose and its subsequent oxidation to CO2 only to a minor degree (30 and 10%, respectively). The absence of external Na+ further depressed the phlorizin effect. Preincubation of the tissue with phlorizin and subsequent washing in part abolished the inhibitory effect. The data suggest that a major portion of the galactose uptake by the tissue proceeds by a mechanism with a low affinity for phlorizin. 4. Efflux studies showed that the wash-out of free galactose from slices was associated with a net decrease of both free and phosphorylated tissue sugar. 5. The above results suggest the possibility that phosphorylation may represent a step in the Na+ -independent, phloretin-sensitive transfer of D-galactose across the antiluminal cell membrane. The participation of intracellular galactokinase and a Zn2+ -activated alkaline phosphatase in the maintenance of the steady state of free and phosphorylated galactose in the cells has been demonstrated.     

Surface carbohydrates of hamster fibroblasts. I. Chemical characterization of surface-labeled glycosphingolipids and aspecific ceramide tetrasaccharide for transformants.

1. Neutral glycosphingolipids of hamster fibroblast NIL cells have been characterized as follows: glucosylceramide, lactosylceramide (betaGall yields 4Glc yields Cer), a digalactosylceramide (alphaGall yields 4betaGal yields Cer), a trihexosylceramide (alphaGall yields 4betaGall yields 4Glc yields Cer), two kinds of ceramide tetrasaccharides (A: alphaGa1NAcl yields 3betaGalNAcl yields 3alphaGall yields 4betaGall yields 1Cer, a new type of Forssman active glycolipid; B: globoside, betaGalNAcl yields 3alphaGall yields 4betaGall yields 4betaGlc yields Cer), and a ceramide pentasaccharide having a classical structure for Forssman antigen (alphaGalNAcl yields 3betaGalNAcl yields 3alphaGall yields 4betaGall yields 4Glc yields Cer). 2. Neutral glycosphingolipids of polyoma virus-transformed NIL cells (NILpy) have been characterized as having an additional ceramide tetrasaccharide which was absent in normal NIL cells. The structure of this specific glycolipid was identified as lacto-N-neotetraosylceramide (betaGall yields 4betaGlc-NAcl yields 3betaGall yields 4Glc yields Cer). Chemical quantities of ceramide tetra- and pentasaccharides in NILpy cells were much lower than in NIL cells. 3. All of these glycolipids, except glucosylceramide and lactosylceramide, were labeled externally by galactose oxidase and tritiated borohydride according to the method previously described (GAHMBERG, C. G, and HAKOMORI, S. (1973) J. Biol. Chem. 248, 4311-4317). The specific activities of the label in glycolipid of NIHpy cells were much greater than that in NIL cells, i.e. reactivity of glycolipids with galactose oxidase in NIHpy cells was much higher than for NIL cells. The surface label in glycolipids was cell cycle-dependent in NIL cells, and a remarkable exposure of a galactosyl residue of a ceramide tetrasaccharide was demonstrated only on the surface of NILpy cells, due to the presence of lacto-N-neotetraosylceramide.     

Differential selectivity of the Escherichia coli cell membrane shifts the equilibrium for the enzyme-catalyzed isomerization of galactose to tagatose

An Escherichia coli galactose kinase gene knockout (DeltagalK) strain, which contains the l-arabinose isomerase gene (araA) to isomerize d-galactose to d-tagatose, showed a high conversion yield of tagatose compared with the original galK strain because galactose was not metabolized by endogenous galactose kinase. In whole cells of the DeltagalK strain, the isomerase-catalyzed reaction exhibited an equilibrium shift toward tagatose, producing a tagatose fraction of 68% at 37 degrees C, whereas the purified l-arabinose isomerase gave a tagatose equilibrium fraction of 36%. These equilibrium fractions are close to those predicted from the measured equilibrium constants of the isomerization reaction catalyzed in whole cells and by the purified enzyme. The equilibrium shift in these cells resulted from the higher uptake and lower release rates for galactose, which is a common sugar substrate, than for tagatose, which is a rare sugar product. A DeltamglB mutant had decreased uptake rates for galactose and tagatose, indicating that a methylgalactoside transport system, MglABC, is the primary contributing transporter for the sugars. In the present study, whole-cell conversion using differential selectivity of the cell membrane was proposed as a method for shifting the equilibrium in sugar isomerization reactions.     

Expression of ApoE gene in Chinese hamster cells with a reversible defect in O-glycosylation. Glycosylation is not required for apoE secretion

The effects of O-glycosylation on the synthesis and secretion of apolipoprotein E (apoE, a glycoprotein with O- but not N-linked sugars) were studied with a UDP-galactose/UDP-N-acetylgalactosamine 4-epimerase-deficient cell mutant (ldlD cells) which expresses a reversible defect in protein O-glycosylation. Under normal culture conditions the mutant ldlD cells cannot add N-acetylgalactosamine (GalNAc) to proteins. GalNAc is the first sugar of mucin-type O-linked oligosaccharides attached to the protein. This O-glycosylation defect is rapidly corrected when GalNAc is added to the culture medium. These cells also require external sources of galactose for the addition of this sugar to O-linked and other oligosaccharides. A bovine papilloma virus-based expression vector for human apoE and the human metallothionein 1A gene were transfected into ldlD cells, and apoE-expressing cell clones resistant to CdCl2 were selected and used in the present studies. The structure and secretion of apoE in these cells were examined by immunoprecipitation and one- and two-dimensional gel electrophoresis and autoradiography. The synthesis, rate, and extent of secretion of apoE were unaffected by O-glycosylation (GalNAc-independent). In the presence of both galactose and GalNAc, multiple apoE isoforms were synthesized in ldlD cells as a result of variation in the extent of sialylation. ApoE sialylation was dependent on the addition of galactose as well as GalNAc to the extracellular medium, suggesting that addition of galactose to the nascent oligosaccharide chains was required for the addition of sialic acid.     

The Agrobacterium tumefaciens virulence gene chvE is part of a putative ABC-type sugar transport operon.

The Agrobacterium tumefaciens virulence determinant ChvE is a periplasmic binding protein which participates in chemotaxis and virulence gene induction in response to monosaccharides which occur in the plant wound environment. The region downstream of the A. tumefaciens chvE gene was cloned and sequenced for nucleotide and expression analysis. Three open reading frames transcribed in the same direction as chvE were revealed. The first two, together with chvE, encode putative proteins of a periplasmic binding protein-dependent sugar uptake system, or ABC-type (ATP binding cassette) transporter. The third open reading frame encodes a protein of unknown function. The deduced transporter gene products are related on the amino acid level to bacterial sugar transporters and probably function in glucose and galactose uptake. We have named these genes gguA, -B, and -C, for glucose galactose uptake. Mutations in gguA, gguB, or gguC do not affect virulence of A. tumefaciens on Kalanchoe diagremontiana; growth on 1 mM galactose, glucose, xylose, ribose, arabinose, fucose, or sucrose; or chemotaxis toward glucose, galactose, xylose, or arabinose.     

Sugar composition of biofilms produced by paper mill bacteria

Biofilms of paper mill bacteria were cultivated in paper mill white water-simulating conditions on glass slides or stainless steel coupons in a laboratory culture system. The sugar content and composition of the biofilms were analysed and compared with the sugar composition of paper mill slimes. Acid methanolysis followed by gas chromatography revealed that Burkholderia was the major biofilm producer in pure culture, producing up to 50 microg of biofilm sugar cm(-2) in 5 days in rich medium and 10 microg in paper mill simulating medium. A mixture of simulated paper mill water with a culture medium yielded more biofilm (100 microg cm(-2)) than either of the media alone, so the biofilm accumulation was not proportional to the available substrate. More biofilm accumulated on stainless steel coupons than on glass slides, and the steel-coupon biofilms contained slightly more uronic acids. The biofilm sugars contained mainly galactose, glucose, mannose, and rhamnose. In paper mill medium, the Burkholderia biofilm contained more galactose and glucose, and less rhamnose, than in rich laboratory medium. The sugar composition of paper mill slimes was quite similar to those of steel-cultured Burkholderia cepacia biofilms. This suggests that Burkholderia cepacia is responsible for much of the slime in the paper mill.     

Binding and spreading of hepatocytes on synthetic galactose culture surfaces occur as distinct and separable threshold responses

Isolated rat hepatocytes bind to synthetic flat polyacrylamide matrices containing covalently attached galactose residues in a sugar-specific and concentration-dependent manner. Cell binding is mediated by the asialoglycoprotein receptor and occurs as a threshold response at or above a critical concentration of sugar in the matrix. Hepatocytes in the presence or absence of serum were able to spread on these synthetic galactose surfaces and were morphologically similar to cells on tissue culture plastic. Cell spreading also occurred as a threshold response but at a much higher critical concentration of sugar than for the cell-binding response. Above the critical concentration for spreading, the area occupied by a cell increased as the sugar concentration increased. By manipulating the galactose content of the matrix, cell spreading and cell binding can be differentiated as independent and separable threshold responses to the extracellular substratum.