The biosynthesis and wall-binding of hemicelluloses in cellulose-deficient maize cells:An example of metabolic plasticity

Cell-suspension cultures(Zea mays L.,Black Mexican sweet corn) habituated to 2,6-dichlorobenzonitrile(DCB) survive with reduced cellulose owing to hemicellulose network modification. We aimed to de fine the hemicellulose metabolism modifications in DCB-habituated maize cells showing a mild reduction in cellulose at different stages in the culture cycle. Using pulse-chase radiolabeling, we fed habituated and non-habituated cultures with [3H]arabinose,and traced the distribution of 3H-pentose residues between xylans, xyloglucans and other polymers in several cellular compartments for 5 h. Habituated cells were slower taking up exogenous [3H]arabinose. Tritium was incorporated into polysaccharide-bound arabinose and xylose residues, but habituated cells diverted a higher proportion of their new [3H]xylose residues into(hetero) xylans at the expense of xyloglucan synthesis. During logarithmic growth, habituated cells showed slower vesicular traf ficking of polymers,especially xylans. Moreover, habituated cells showed a decrease in the strong wall-binding of all pentose-containing polysaccharides studied; correspondingly, especially in log phase cultures, habituation increased the proportion of 3H-hemicelluloses([3H]xylans and [3H]xyloglucan) sloughed into the medium. These findings could be related to the cel walls' cellulose-deficiency, and consequent reduction in binding sites for hemicelluloses; the data could also re fl ect the habituated cells' reduced capacity to integrate arabinox ylans by extra-protoplasmic phenolic cross-linking, as well as xyloglucans, during wall assembly.     

Restructuring of wall-bound xyloglucan by transglycosylation in living plant cells

Xyloglucan endotransglycosylases (XETs) cleave and then re-join xyloglucan chains and may thus contribute to both wall-assembly and wall-loosening. The present experiments demonstrate the simultaneous occurrence in vivo of two types of interpolymeric transglycosylation: "integrational" (in which a newly secreted xyloglucan reacts with a previously wall-bound one) and "restructuring" (in which one previously wall-bound xyloglucan reacts with another). Xyloglucans synthesised by cultured rose (Rosa sp.) cells in "heavy" or "light" media (with [13C,2H]glucose or [12C,1H]glucose, respectively) had buoyant densities of 1.643 and 1.585 g ml-1, respectively, estimated by isopycnic centrifugation in caesium trifluoroacetate. To detect transglycosylation, we shifted heavy rose cells into light medium, then supplied a 2-h pulse of L-[1-3H]arabinose. Light [3H]xyloglucans were thus secreted into heavy, non-radioactive walls and chased by light, non-radioactive xyloglucans. At 2 h after the start of radiolabelling, the (neutral) [3H]xyloglucans were on average 29% heavy, indicating molecular grafting during integrational transglycosylation. The [3H]xyloglucans then gradually increased in density until, by 11 h, they were 38% heavy. This density increase suggests that restructuring transglycosylation reactions occurred between the now wall-bound [3H]xyloglucan and other (mainly older, i.e. heavy) wall-bound non-radioactive xyloglucans. Brefeldin A (BFA), which blocked xyloglucan secretion, did not prevent the increase in density of wall-bound [3H]xyloglucan (2-11 h). This confirms that restructuring transglycosylation occurred between pairs of previously wall-bound xyloglucans. After 7 d in BFA, the 3H was in hybrid xyloglucans in which on average 55% of the molecule was heavy. Exogenous xyloglucan oligosaccharides (competing acceptor substrates for XETs) did not affect integrational transglycosylation whereas they inhibited restructuring transglycosylation. Possible reasons for this difference are discussed. This is the first experimental evidence for restructuring transglycosylation in vivo. We argue that both integrational and restructuring transglycosylation can contribute to both wall-assembly and -loosening.     

Kinetics of Integration of Xyloglucan into the Walls of Suspension-Cultured Rose Cells

To study the kinetics of synthesis, wall-binding and degradationof xyloglucan, we incubated suspension-cultured rose cells for0–5–24 h in L-[1-³H]arabinose. >95% of the [³H]arabinosewas taken up within 2 h. UDP-Pentoses were maximally labelledwithin 0–5 h and had lost most of their ³H by 2 h afterthe addition of [³H]arabinose. Therefore, the 24 h experimentresembled a pulse-chase rgime. The [³H]xyloglucan formed wasfractionated into four cellular pools [detergent-extractable(interpreted as cytoplasmic), and guanidinium thiocyanate-,06 M NaOH- and 60 M NaOH-extractable (interpreted as progressivelymore firmly wall-bound)]; soluble extracellular xyloglucan wascollected as a fifth pool. All five pools of xyloglucan hadstarted accumulating ³H at their respective maximal rates by     

Cell wall changes in immature Asparagus stem tissue after excision.

Cell wall material (CWM) was prepared from sections of fresh and aerobically-stored asparagus (Asparagus officinalis, L. cv. Connovor Collossus) stems. Polymers were solubilized from the CWM by successive extraction with cyclohexane-trans-1,2-diamine-N N N' N'-tetraacetate (CDTA), Na2CO3 and KOH to leave the alpha-cellulose residue which contained a significant amount of cross-linked pectic polysaccharides. The polymers were fractionated by anion-exchange chromatography and selected fractions were subjected to methylation analysis. The storage-related decrease in (1-4)-linked Galp was detected in all the fractions rich in pectic polysaccharides, particularly in the CDTA, Na2CO3, 0.5 M KOH fractions and alpha-cellulose residue. A smaller decrease in Araf was also observed. This was mainly due to a decrease in (1-5)-linked Araf in the Na2CO3-1-soluble polymers, and terminal Araf in the alpha-cellulose residue. There was evidence for the occurrence of significant amounts of complexes containing pectic polysaccharides and xylans having a relatively low degree of polymerization in the dilute alkali-soluble polymers, and some of these contained phenolic compounds; the storage-induced increase in (1-4)-linked Xylp was confined to these polymers. Interestingly, no free acidic xylans could be detected in the 1 M and 4 M KOH-soluble polymers; instead, the bulk of the hemicellulosic polysaccharides appeared to be mixtures of xyloglucans and xylans in which the ratio of xyloglucan to xylan increased with increasing strength of alkali used for extraction of the polymers. The non-degradative extraction and fractionation procedures revealed heterogeneity in pectic polysaccharides, pectic polysaccharide-xylan complexes and xyloglucans in close association with xylans. The possible relationship between pectic polysaccharide-xylan-phenolic complexes and the onset of lignification in maturing tissues is discussed.     

Xyloglucan−pectin linkages are formed intra-protoplasmically,contribute to wall-assembly,and remain stable in the cell wall

We tested two hypotheses for the mechanism by which xyloglucan–pectin covalent bonds are formed in Arabidopsis cell cultures. Hypothesis 1 proposed hetero-transglycosylation, with xyloglucan as donor substrate and a rhamnogalacturonan-I (RG-I) side-chain as acceptor. We looked for enzyme activities that catalyse this reaction using α-(1→5)-l-[³H]arabino- or β-(1→4)-d-[³H]galacto-oligosaccharides as model acceptor substrates. The ³H-oligosaccharides were supplied (with or without added xyloglucans) to living Arabidopsis cell-cultures, permeabilised cells, cell-free extracts, or four authentic XTHs. No hetero-transglycosylation occurred. Therefore, we cannot support hypothesis 1. Hypothesis 2 proposed that some xyloglucan is manufactured de novo as a side-chain on RG-I. To test this, we pulse-labelled Arabidopsis cell-cultures with [³H]arabinose and monitored the radiolabelling of anionic (pectin-bonded) xyloglucan, which was resolved from free xyloglucan by ion-exchange chromatography. [³H]Xyloglucan–pectin complexes were detectable <4 min after [³H]arabinose feeding, which is shorter than the transit-time for polysaccharide secretion, indicating that xyloglucan–pectin bonds were formed intra-protoplasmically. Thereafter, the proportion of the wall-bound [³H]xyloglucan that was anionic remained almost constant at ∼50% for ≥6 days, showing that the xyloglucan–pectin bond was stable in vivo. Some [³H]xyloglucan was rapidly sloughed into the medium instead of becoming wall-bound. Only ∼30% of the sloughed [³H]xyloglucan was anionic, indicating that bonding to pectin promoted the integration of xyloglucan into the wall. We conclude that ∼50% of xyloglucan in cultured Arabidopsis cells is synthesised on a pectic primer, then secreted into the apoplast, where the xyloglucan–pectin bonds are stable and the pectic moiety aids wall-assembly.     

Xyloglucan oligosaccharides cause cell wall loosening by enhancing xyloglucan endotransglucosylase/hydrolase activity in azuki bean epicotyls

Addition of xyloglucan-derived oligosaccharides shifted the wall-bound xyloglucans to a lower molecular mass distribution and increased the cell wall extensibility of the native epidermal tissue strips isolated from azuki bean (Vigna angularis) epicotyls. To ascertain the mechanism of oligosaccharide function, we examined the action of a xyloglucan endotransglucosylase/hydrolase (XTH) showing both endotransglucosylase and endohydrolase activities, isolated from azuki bean epicotyl cell walls, in the presence of xyloglucan oligosaccharides. The addition of xyloglucan oligosaccharides enhanced the xyloglucan-degrading activity of XTH against isolated xyloglucan substrates. When the methanol-fixed epidermal tissue strips were incubated with XTH, the molecular mass of wall-bound xyloglucans was decreased and the cell wall extensibility increased markedly in the presence of the oligosaccharides. These results suggest that xyloglucan oligosaccharides stimulate the degradation of xyloglucans by enhancing the XTH activity within the cell wall architecture, thereby increasing the cell wall extensibility in azuki bean epicotyls.     

In vivo Binding of Gibberellin A(1) in Dwarf Pea Epicotyls

Binding of [(3)H]gibberellin A(1) (GA(1)) to extracts of dwarf pea epicotyls was investigated using sliced pea epicotyls (0.5-1.0 millimeter thick) that had been incubated in a solution containing [(3)H]GA(1) at 0 C for 3 days. Gel filtration of a 100,000g supernatant indicated binding to a high (HMW) and an intermediate molecular weight (IMW) fraction with estimated molecular weights of 6 x 10(5) daltons and 4 to 7 x 10(4) daltons, respectively. The bound (3)H-activity was [(3)H]GA(1) and not a metabolite as deduced by thin layer chromatography. The bound label did not sediment during centrifugation at 100,000g for 2 hours; also, binding was not disrupted after treatment of a combined HMW and IMW fraction with DNase, RNase, or phospholipase A or C, but it was disrupted by protease or heat treatment. These facts suggest that binding of [(3)H]GA(1) was occurring to a soluble protein(s). [(3)H]GA(1) bound to a combined HMW and IMW fraction was not susceptible to changes in pH, nor could it be exchanged with a variety of GAs tested under in vitro conditions. Under in vivo equilibrium conditions, biologically active GAs, such as GA(1), GA(3), GA(4), GA(5), GA(7), and keto GA(1), could reduce the level of [(3)H]GA(1) binding, whereas inactive GAs, such as iodo GA(1) methyl ester, GA(8), GA(13), GA(26), and non-GAs, such as (+/-)abscisic acid, had no effect. By varying the concentration of [(3)H]GA(1) in the incubation medium, the specific binding of [(3)H]GA(1) appeared to be due to two classes of binding sites having estimated K(d) of 6 x 10(-8) molar and 1.4 x 10(-6) molar. The concentrations of the two sites were estimated to be 0.45 picomole per gram and 4.04 picomoles per gram on a fresh weight and 0.1 picomole per milligram and 0.9 picomole per milligram on a soluble protein basis, respectively.     

Trimming and solubilization of xyloglucan after deposition in the walls of cultured rose cells

A pulse-chase technique involving the in vivo feeding of L-[1-³H]arabinoseto suspension-cultured rose (Rosa) cells at 4 d and 9 d aftersubculture (fast- and slow-growing, respectively) was used tocreate a population of [³H]xyloglucan molecules and to followtheir subsequent fate. The weight-average relative molecu larmass (M_w) of [³H]xyloglucan freshly deposited in the cell wallwas 160 000 and 240 000 in the fast- and slow-growing cells,respectively. The wall-bound [³H]xyloglucan of both culturesunderwent a decrease in M_w of 40 000 during the first 2 d afterthe pulse-labelling. At the same time, 20–30% of the initially-deposited[³H]xyloglucan disappeared from the cell wall, and a similaramount appeared in solution in the culture medium. Its failureto remain bound to the cell wall and its low M_w (39 000) indicatedthat this soluble extracellular ( was derived from partial degradationof segments of wall-bound xyloglucan that were not directlyhydrogen-bonded to microfibrils (‘loose ends’ and‘tethers’). The possible enzymic basis and biologicalroles of the degradation are discussed. Key words: Cell expansion, cell wall, hemicellulose, sloughing, xyloglucan     

Cyclic 3', 5'-AMP relay dictyostelium discoideum. V. Adaptation of the cAMP signaling response during cAMP stimulation

In dictyostelium discoideum, extracellular cAMP activates adenylate cyclase, which leads to an increase in intracellular cAMP and the rate of cAMP secretion. The signaling response to a constant cAMP stimulus is terminated after several minutes by an adaptation mechanism. The time- course of adaptation stimuli of 10(-6) or 10(-7) M cAMP was assessed. We used a perfusion technique to deliver defined cAMP stimuli to [(3)H]adenosine-labeled amoebae and monitored their secretion of [(3)H]cAMP. Amoebae were pretreated with 10(-6) or 10(-7) M cAMP to periods of 0.33-12 minutes, and then immediately given test stimuli of 10(-8) M to 2.5 x 10(-7) M cAMP. The response to a given test stimulus was progressively attenuated and finally extinguished as the duration of the pretreatment stimulus increased. During concentration of the test stimulus. The responses to test stimuli of 10(-8), 5 x 10(-8), 10(-7), or 2.5 x 10(-7) M cAMP were extinguished after approximately 1, 2.25,2.5, and 10 min, respectively. 1.5 min of stimulation with 10(-7) M cAMP was necessary to extinguish the response of a test stimulus of 10(-8) M cAMP. Our data suggest that adaptation begins within 20 s of stimulation, rises rapidly for approximately 2.5 min, and reaches a plateau after approximately 10 min. The absolute rate of rise was faster during pretreatment with 10(-6) than with 10(-7) M cAMP. These results support a working hypothesis in which the occupancy of surface cAMP receptors leads to changes in two opposing cellular processes, excitation and adaptation, that control the activity of D. discoideum adenylate cyclase.     

Beta-Xylosidases and a nonspecific wall-bound beta-glucosidase of the yeast Cryptococcus albidus

Cryptococcus albidus grown on wood xylans possesses a soluble intracellular beta-xylosidase (EC 3.2.1.37) as an additional constituent of the xylan-degrading enzyme system of this yeast. The enzyme attacks linear 1,4-beta-xylooligosaccharides in an exo-fashion, liberating xylose from the non-reducing ends. The activity of the enzyme increases in the cells during growth on xylan and incubation with xylobiose or methyl beta-D-xylopyranoside which are the best inducers of extracellular beta-xylanase (EC 3.2.1.8). Various alkyl-,alkyl-1-thio- and aryl beta-D-xylopyranosides were excellent inducers of a different beta-xylosidase of Cryptococcus albidus. This enzyme is localized outside the plasma membrane and is principally associated with cell walls. Unlike the soluble intracellular beta-xylosidase, the wall-bound enzyme does not hydrolyze xylooligosaccharides. Evidence has been obtained that beta-xylosidase activity in the cell walls is not due to the presence of a specific aryl beta-xylosidase, but is exhibited by a nonspecific beta-glucosidase (EC 3.2.1.21) inducible by beta-D-xylopyranosides. The ratio of beta-glucosidase and beta-xylosidase activity in the cells and isolated cell walls from yeast induced by various beta-xylopyranosides and beta-glucopyranosides was very similar. Both wall-bound activities were inhibited in a similar pattern by inhibitors of beta-glucosidases, 1,5-gluconolactone and nojirimycin. This bifunctional enzyme does not bear any relationship to the utilization of xylans in Cryptococcus albidus.     

Asparagine biosynthesis in soybean nodules

Two auxin-induced endo-1,4-β-glucanases (EC 3.2.1.4) were purified from pea (Pisum sativum L. var. Alaska) epicotyls and used to degrade purified pea xyloglucan. Hydrolysis yielded nonasaccharide (glucose/xylose/galactose/fucose, 4:3:1:1) and heptasaccharide (glucose/xylose, 4:3) as the products. The progress of hydrolysis, as monitored viscometrically (with amyloid xyloglucan) and by determination of residual xyloglucan-iodine complex (pea) confirmed that both pea glucanases acted as endohydrolases versus xyloglucan. K_m values for amyloid and pea xyloglucans were approximately the same as those for cellulose derivatives, but V_max values were lower for the xyloglucans. Auxin treatment of epicotyls in vivo resulted in increases in net deposits of xyloglucan and cellulose in spite of a great increase (induction) of endogenous 1,4-β-glucanase activity. However, the average degree of polymerization of the resulting xyloglucan was much lower than in controls, and the amount of soluble xyloglucan increased. When macromolecular complexes of xyloglucan and cellulose (cell wall ghosts) were treated in vitro with pea 1,4-β-glucanase, the xyloglucan component was preferentially hydrolyzed and solubilized. It is concluded that xyloglucan is the main cell wall substrate for pea endo-1,4-β-glucanase in growing tissue.     

Co-operative binding of triplicate carbohydrate-binding modules from a thermophilic xylanase

Family 6 carbohydrate-binding modules were amplified by polymerase chain reaction (PCR) from Clostridium stercorarium strain NCIB11754 genomic DNA as a triplet. Individually, these modules bound to xylooligosaccharides and cellooligosaccharides with affinities varying from approximately 3 x 10(3) M(-1) to approximately 1 x 10(5) M(-1). Tandem and triplet combinations of these modules bound co-operatively to soluble xylan and insoluble cellulose to give approximately 20- to approximately 40-fold increases in affinity relative to the individual modules. This co-operativity was an avidity effect resulting from the modules within the tandems and triplet interacting simultaneously with proximal binding sites on the polysaccharides. This occurred by both intrachain and interchain interactions. The duplication or triplication of modules appears to be linked to the growth temperature of the organism; co-operativity in these multiplets may compensate for the loss of affinity at higher temperatures.     

Binding specificity and thermodynamics of a family 9 carbohydrate-binding module from Thermotoga maritima xylanase 10A

The C-terminal family 9 carbohydrate-binding module of xylanase 10A from Thermotoga maritima (CBM9-2) binds to amorphous cellulose, crystalline cellulose, and the insoluble fraction of oat spelt xylan. The association constants (K(a)) for adsorption to insoluble polysaccharides are 1 x 10(5) to 3 x 10(5) M(-1). Of the soluble polysaccharides tested, CBM9-2 binds to barley beta-glucan, xyloglucan, and xylan. CBM9-2 binds specifically to the reducing ends of cellulose and soluble polysaccharides, a property that is currently unique to this CBM. CBM9-2 also binds glucose, xylose, galactose, arabinose, cellooligosaccharides, xylooligosaccharides, maltose, and lactose, with affinities ranging from 10(3) M(-1) for monosaccharides to 10(6) M(-1) for disaccharides and oligosaccharides. Cellooligosaccharides longer than two glucose units do not bind with improved affinity, indicating that cellobiose is sufficient to occupy the entire binding site. In general, the binding reaction is dominated by favorable changes in enthalpy, which are partially compensated by unfavorable entropy changes.     

Fractionation of hemicelluloses from maize cell walls with increasing concentrations of alkali

Hemicelluloses were solubilized from depectinated walls of maize coleoptiles and leaves with increasing concentrations of alkali to yield three major fractions of polymers. A highly-substituted glucuronoarabinoxylan released by dilute alkali from walls of coleoptiles was present only in very small amounts in the walls of the leaves. The stepwise extractions with increasing concentrations of alkali resolved a relatively unbranched xylan from a mixture of mixed-linked glucan, xyloglucan and additional xylan from walls of young leaves. Delignification in acidic sodium chlorite solubilized a small amount of substituted xylan from walls of both coleoptiles and leaves, and rendered about one-half of the unextracted hemicellulose soluble in only 0.02 M potassium hydroxide solution. Delignification prevented the detection of highly-substituted xylans released by dilute alkali.     

Role of human skin in the photodecomposition of bilirubin

1. Human skin epithelium and human skin were found to absorb both free bilirubin and serum-bound bilirubin from an aqueous buffered medium. The serum-bound bilirubin thus absorbed was readily released when human skin epithelium or human skin were transferred to media containing no bilirubin. 2. The K(m) values for serum-bound bilirubin were 1.8x10(-3)m and 2.2x10(-3)m respectively for human skin epithelium and human skin; corresponding K(m) values for free bilirubin were 3.0x10(-4)m and 5x10(-4)m. The V(max.) for bound and free bilirubin was of the same magnitude, the apparent V(max.) being 1.0 and 1.66mumol/g of tissue for human skin epithelium and human skin respectively. 3. When human skin that had acquired a yellow tinge by absorbing bilirubin was incubated in a buffered medium and exposed to a mercury-vapour light, the yellow colour disappeared and decomposition products of bilirubin accumulated in the medium. 4. Experiments with [(3)H]bilirubin indicated that the pigment absorbed by skin was photo-oxidized to products that were soluble in water and the quantity and number of such products increased with the time of exposure of human skin to the light-source. Under similar conditions [(3)H]bilirubin alone in buffered medium was also oxidized and gave products which by paper chromatography appeared to be different from those released by human skin that had absorbed bilirubin. 5. The results suggest that by virtue of its large surface area human skin can act as a matrix for the degradative action of light on bilirubin.     

Presynaptic alpha2-receptors regulate reverse Na+/Ca2+-exchange and transmitter release in Na+-loaded peripheral sympathetic nerves

Electrical depolarisation-(2 Hz, 1 ms)-induced [3H]noradrenaline ([3H]NA) release has been measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3 x 10(-5) M; corticosterone, 5 x 10(-5) M). Substitution of most of the external Na+ by Li+ (113 mM; [Na+]0: 25 mM) slightly potentiated the axonal stimulation-evoked release of [3H]NA in a tetrodotoxin (TTX, 10(-7) M) sensitive manner. The reverse Na+/Ca2+-exchange inhibitor KB-R7943 (3 x 10(-5) M) failed to inhibit the stimulation-evoked release of [3H]NA, but increased the resting outflow of neurotransmitter. The 'N-type' voltage-sensitive Ca2+-channel (VSCC) blocker omega-conotoxin (omega-CgTx) GVIA (10(-8) M) significantly and irreversibly inhibited the release of [3H]NA on stimulation (approximately 60-70%). The 'residual release' of NA was abolished either by TTX or by reducing external Ca2+ from 2.5 to 0.25 mM. The 'residual release' of NA was also blocked by the non-selective VSCC-blocker neomycin (3 x 10(-3) M). Correlation was obtained between the extent of VSCC-inhibition and the transmitter release-enhancing effect of presynaptic alpha2-receptor blocker yohimbine (3 x 10(-7) M). When the release of [3H]NA was blocked by omega-CgTx GVIA plus neomycin, yohimbine was ineffective. Inhibition of the Na+-pump by removal of K+ from the external medium increased both the resting and the axonal stimulation-evoked release of [3H]NA in the absence of functioning VSCCs (i.e., in the presence of neomycin and after omega-CgTx treatment). Under these conditions the stimulation-evoked release of NA was abolished either by TTX or by external Ca2+-removal (+1 mM EGTA). Similarly, external Li+ (113 mM) or the reverse Na+/Ca2+ exchange blocker KB-R7943 (3 x 10(-5) M) significantly inhibited the stimulation-induced transmitter release in 'K+-free' solution. KB-R7943 decreased the resting outflow of NA as well. Under conditions in which the Na+-pump was inhibited in the absence of functioning VSCCs, yohimbine (3 x 10(-7) M) further enhanced the release of neurotransmitter, while l-noradrenaline (l-NA, 10(-6) M), an agonist of presynaptic alpha2-receptors, inhibited it. The yohimbine-induced enhancement of NA-release was abolished by Li+-substitution and significantly inhibited by KB-R7943 application. It is concluded that after blockade of VSCCs brief depolarising pulses may reverse Na+/Ca2+-exchange and release neurotransmitter in Na+-loaded sympathetic nerves. Further, similar to that of VSCCs, the reverse Na+/Ca2+-exchange may also be regulated by presynaptic alpha2-receptors.     

Purification and Biophysical Properties of Acute Haemorrhagic Conjunctivitis Virus

The buoyant density of acute haemorrhagic conjunctivitis virions labeled with either [(3)H]uridine or [(3)H]leucine was 1.34 g/ml in CsCl and 1.25 g/ml in sucrose. RNA extracted from the virions gave a sedimentation coefficient of approximately 34S in sucrose, and was found to be sensitive to RNase. Molecular weight of RNA was calculated to be 2.5 x 10(6) using poliovirus RNA for reference.     

Putrescine uptake and release by a normal rat small intestine crypt cell line, IEC-6

IEC-6 cells were cultured on permeable filter inserts with separate access to the apical and basolateral sides. [3H]Putrescine uptake favored the apical side and its release (in Earle's balanced salt solution containing 0.1% bovine serum albumin) was six times greater in the apical-to-basolateral than in the basolateral-to-apical direction. Release in DMEM did not show this preference. The uptake of [3H]putrescine was stimulated approximately 1.3 times the basal level by 10 mM asparagine (ASN) or 5% dialyzed fetal bovine serum whether the [3H]putrescine was added at a concentration of 1 or 100 nM. The increased uptake was maintained for up to 6 h. When [3H]putrescine was removed after 4 h of uptake, the cells continued to release it into the medium on both sides for up to 4 h. Stimulated cells released only 50% as much as unstimulated cells. Unlabeled putrescine reduced the uptake of [3H]putrescine with an IC50 of 1.81 x 10(-6) M (r = 0.9476) and 1.02 x 10(-6) M (r = 0.9967) for unstimulated and ASN-stimulated cells, respectively. When the intracellular putrescine was reduced by difluoromethylornithine, the uptake of [3H]-putrescine was not changed, but its release was inhibited. Sodium was not required for [3H]putrescine uptake or release. Although the stimulated cells attained intracellular levels of [3H]putrescine which, if expressed as concentration based on cell volume, were up to 500 times the original extracellular concentration, a true concentration gradient could not be proven because 85% of the [3H]putrescine was probably bound to polyanions as shown by butanol extraction.     

Characterization of O-acetyl-(4-O-methylglucurono)xylan isolated from birch and beech

The structures of water-soluble birch and beech xylans, extracted from holocellulose using dimethyl sulfoxide, were determined employing 1H and 13C NMR spectroscopy together with chemical analysis. These polysaccharides were found to be O-acetyl-(4-O-methylglucurono)xylans containing one 4-O-methylglucuronic acid substituent for approximately every 15 D-xylose residues. The average degree of acetylation of the xylose residues in these polymers was 0.4. The presence of the structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1--> was demonstrated. Additional acetyl groups were present as substituents at C-2 and/or C-3 of the xylopyranosyl residues. Utilizing size-exclusion chromatography in combination with mass spectroscopy, the weight-average molar masses (and polydispersities) were shown to be 8000 (1.09) and 11,100 (1.08) for birch and beech xylan, respectively.     

Biochemical characterization of rab proteins from Bombyx mori

The small GTPases known as Rab proteins are key regulators of membrane trafficking. We used RT-PCR to isolate cDNA clones of insect-specific Rab proteins (BRabN1 and BRabN2) showing low homology with known Rab proteins from other animals, from mRNA of Bombyx mori. These 2 Rabs were produced in Escherichia coli and purified. BRabN1 bound [(3)H]-GDP and [(35)S]-GTPgammaS with dissociation constants of 0.087 x 10(-6) M and 1.02 x 10(-6) M, respectively, whereas those of BRabN2 were 0.546 x 10(-6) M and 1.02 x 10(-6) M, respectively. Binding of [(35)S]-GTPgammaS to BRabN1 and N2 was inhibited by GDP and GTP. The GTP-hydrolysis activities of BRabN1 and N2 were 154 and 35.5 mmol/min/mole, respectively, and bound [(35)S]-GTPgammaS was exchanged efficiently with GTP. BRabN1 also showed ATPase activity and exchange of [(35)S]-GTPgammaS with ATP. Monoclonal antibodies against BRabN1 and N2 did not recognize any other Rab proteins, and Western blotting using the anti-BRabN1 antibody revealed a single band in the testis of B. mori. These results suggest that BRabN1 and N2 of B. mori bind GTP, convert from the GTP-bound state to the GDP-bound state by intrinsic GTP hydrolysis activity, and return to the GTP-bound state with the exchange, and that BRabN1 is specifically expressed in testis. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.