Utility of pentose colorimetric assay for the purification of potato lectin, an arabinose-rich glycoprotein

Potato lectin (Solanum tuberosum agglutinin, STA) is an unusual glycoprotein containing approximately 50% carbohydrates by weight. Of the total carbohydrates, 92% is contributed by L-arabinose, which are O-linked to hydroxyproline residues. The ferric chloride-orcinol assay (Bial’s test), which is specific for pentoses has so far been used only for the determination of free pentoses in biological samples. However, this colorimetric assay has not been used for the detection of pentoses in bound form as it occurs in Solanaceae lectins (potato, tomato, and Datura lectins). Utilizing the pentose colorimetric assay for monitoring the presence of potato lectin, a simpler and shorter procedure for the purification of this lectin from potato tubers has been developed. The yield of potato lectin (1.73 mg per 100 g potato tuber) is twice compared to the yields reported in earlier procedures. Although potato lectin is well known for its specificity to free trimers and tetramers of N-acetyl-D-glucosamine (GlcNAc), it possesses a similar specificity to the core (GlcNAc)₂ of N-linked glycoproteins. The utilization of the pentose assay in the purification of arabinose-rich lectins/agglutinins obviates the necessity for the use of agglutination assay in the various purification steps. The pentose assay appears to be a simple and convenient colorimetric assay for detecting any pentose-rich glycoprotein in plant extracts. The utility of the pentose assay appears to have a significant potential in the detection of hydroxyproline-rich glycoproteins (HRGPs), which are generally O-arabinosylated.     

Lactosyl derivatives function in a rat model of severe burn shock by acting as antagonists against CD11b of integrin on leukocytes

Severe burn shock remains an unsolved clinical problem with urgent needs to explore novel therapeutic approaches. In this study, the in vivo bioactivity of a series of synthetic lactosyl derivatives (oligosaccharides) was assessed on rats with burn shock to elucidate the underlying mechanisms. Administration of An-2 and Gu-4, two lactosyl derivatives with di- and tetravalent β-d-galactopyranosyl-(1-4)-β-d-glucopyranosyl ligands, significantly prolonged the survival time (P < 0.05 vs. saline), stabilized blood pressure and ameliorated the injuries to vital organs after burn. Flow chamber assay displayed that An-2 and Gu-4 markedly decreased the adhesion of leukocytes to microvessel endothelial cells. Competitive binding assay showed that a CD11b antibody significantly interrupted the interaction of An-2 and Gu-4 with leukocytes from rats with burn shock. With fluorescent microscopy, we further found that the oligosaccharides were selectively bound to leukocytes and with a colocalization of CD11b on the cell membrane. Interestingly, the lectin domain-deficient form of CD11b failed to bind with An-2 and Gu-4. The results suggest that both An-2 and Gu-4 significantly inhibit the adhesion of leukocytes to endothelial cells by binding to CD11b and thereby exert protective effects on severe burn shock. Zhihui Zhao and Qing Li contributed equally to this paper.     

Oral supplementation with l-aspartate and l-glutamate inhibits atherogenesis and fatty liver disease in cholesterol-fed rabbit

Previous studies have shown that dietary supplementation with l-aspartate and l-glutamate inhibits fatty streak initiation in cholesterol-fed rabbit. The present study investigates the role of dicarboxylic amino acids on the progression of fatty streaks and the development of fatty liver disease, which were caused in New Zealand White rabbits after a 0.5% w/w cholesterol diet for 7 weeks. A group of animals additionally received a combination of 12.5 mM l-aspartate and 12.5 mM l-glutamate per day through drinking water. Total cholesterol (TC), high-density lipoproteins cholesterol (HDLC), non-HDLC and triacylglycerol (TAG) concentrations were measured in plasma. Serum gamma-glutamyl transferase (γ-GT), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were also determined. At the end of dietary intervention, animals were sacrificed. Aortic, hepatic and brain lesions were evaluated after staining with hematoxylin and eosin. Supplementation with dicarboxylic amino acids inhibited the progression of aortic intima thickness (P < 0.05) and the development of liver lesions (P < 0.05). TC, non-HDLC and TAG were similarly increased in both cholesterol-fed groups. Serum γ-GT and AST activities elevated during the study in all cholesterol-fed animals but the elevation of γ-GT was milder and significantly lower in rabbits treated with l-aspartate and l-glutamate (P < 0.05). ALT activity was not affected by cholesterol feeding. In conclusion, oral supplementation with l-aspartate and l-glutamate inhibits the progression of atherogenesis and the development of fatty liver disease in the animal model of cholesterol-fed rabbit. The beneficial effects of dicarboxylic amino acids reflect the limited elevation of serum γ-GT activity.     

d-lactic acid production from cellooligosaccharides and β-glucan using l-LDH gene-deficient and endoglucanase-secreting Lactobacillus plantarum

In order to achieve direct fermentation of an optically pure d-lactic acid from cellulosic materials, an endoglucanase from a Clostridium thermocellum (CelA)-secreting plasmid was introduced into an l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (∆ldhL1) bacterial strain. CelA expression and its degradation of β-glucan was confirmed by western blot analysis and enzyme assay, respectively. Although the CelA-secreting ∆ldhL1 assimilated cellooligosaccharides up to cellohexaose (although not cellotetraose), the main end product was acetic acid, not lactic acid, due to the conversion of lactic acid to acetic acid. Cultivation under anaerobic conditions partially suppressed this conversion resulting in the production of 1.27 g/l of D-lactic acid with a high optical purity of 99.5% from a medium containing 2 g/l of cellohexaose. Subsequently, D-lactic acid fermentation from barley β-glucan was carried out with the addition of Aspergillus aculeatus β-glucosidase produced by recombinant Aspergillus oryzae and 1.47 g/l of D-lactic was produced with a high optical purity of 99.7%. This is the first report of direct lactic acid fermentation from β-glucan and a cellooligosaccharide that is a more highly polymerized sugar than cellotriose.     

ɛ-Poly-<Emphasis Type="SmallCaps">l</Emphasis>-lysine producer, <Emphasis Type="Italic">Streptomyces albulus</Emphasis>, has feedback-inhibition resistant aspartokinase

Streptomyces albulus NBRC14147 produces ɛ-poly-l-lysine (ɛ-PL), which is an amino acid homopolymer antibiotic. Despite the commercial importance of ɛ-PL, limited information is available regarding its biosynthesis; the l-lysine molecule is directly utilized for ɛ-PL biosynthesis. In most bacteria, l-lysine is biosynthesized by an aspartate pathway. Aspartokinase (Ask), which is the first enzyme in this pathway, is subject to complex regulation such as through feedback inhibition by the end-product amino acids such as l-lysine and/or l-threonine. S. albulus NBRC14147 can produce a large amount of ɛ-PL (1–3 g/l). We therefore suspected that Ask(s) of S. albulus could be resistant to feedback inhibition to provide sufficient l-lysine for ɛ-PL biosynthesis. To address this hypothesis, in this study, we cloned the ask gene from S. albulus and investigated the feedback inhibition of its gene product. As predicted, we revealed the feedback resistance of the Ask; more than 20% relative activity of Ask was detected in the assay mixture even with extremely high concentrations of l-lysine and l-threonine (100 mM each). We further constructed a mutated ask gene for which the gene product Ask (M68V) is almost fully resistant to feedback inhibition. The homologous expression of Ask (M68V) further demonstrated the increase in ɛ-PL productivity.     

Fagopyritol B1, O-α-D-galactopyranosyl-(1→2)-D-chiro-inositol, a galactosyl cyclitol in maturing buckwheat seeds associated with desiccation tolerance

O-α-D-Galactopyranosyl-(1→2)-D-chiro-inositol, herein named fagopyritol B1, was identified as a major soluble carbohydrate (40% of total) in buckwheat (Fagopyrum esculentum Moench, Polygonaceae) embryos. Analysis of hydrolysis products of purified compounds and of the crude extract led to the conclusion that buckwheat embryos have five α-galactosyl D-chiro-inositols: fagopyritol A1 and fagopyritol B1 (mono-galactosyl D-chiro-inositol isomers), fagopyritol A2 and fagopyritol B2 (di-galactosyl D-chiro-inositol isomers), and fagopyritol B3 (tri-galactosyl D-chiro-inositol). Other soluble carbohydrates analyzed by high-resolution gas chromatography included sucrose (42% of total), D-chiro-inositol, myo-inositol, galactinol, raffinose and stachyose (1% of total), but no reducing sugars. All fagopyritols were readily hydrolyzed by α-galactosidase (EC 3.2.1.22) from green coffee bean, demonstrating α-galactosyl linkage. Retention time of fagopyritol B1 was identical to the retention time of O-α-D-galactopyranosyl-(1→2)-D-chiro-inositol from soybean (Glycine max (L.) Merrill, Leguminosae), suggesting that the α-ga-lactosyl linkage is to the 2-position of D-chiro-inositol. Accumulation of fagopyritol B1 was associated with acquisition of desiccation tolerance during seed development and maturation in planta, and loss of fagopyritol B1 correlated with loss of desiccation tolerance during germination. Embryos of seeds grown at 18 °C, a condition that favors enhanced seed vigor and storability, had a sucrose-to-fagopyritol B1 ratio of 0.8 compared to a ratio of 2.46 for seeds grown at 25 °C. We propose that fagopyritol B1 facilitates desiccation tolerance and storability of buckwheat seeds. Received: 21 May 1997 / Accepted: 5 June 1997     

C-reactive protein promotes adhesion of monocytes to endothelial cells via NADPH oxidase-mediated oxidative stress

Enhanced monocyte adhesion to endothelial cells is an early event in atherogenesis. It has been shown that C‐reactive protein (CRP) plays a key role in atherogenesis. Here, we investigated the effects of CRP on monocyte‐endothelial cell adhesion and tested the hypothesis that NADPH oxidase (NOX)‐mediated oxidative stress might play a key role in CRP‐induced monocyte‐endothelial cell adhesion. Firstly, 36 patients with carotid intima‐media thickness (IMT) incrassation and 34 controls were enrolled in this study. The levels of glucose, lipids, CRP, monocyte chemotractant protein (MCP‐1), malondialdehyde (MDA), and protein carbonylation were analyzed. The results showed that carotid IMT was associated with abnormal lipid metabolism, including elevated CRP, triglycerides (TG) (P < 0.01) and decreased high density lipoprotein (HDL) level (P < 0.05). The levels of CRP and MCP‐1 in patients with carotid IMT incrassation were increased compared with the controls (P < 0.01). Moreover, patients with carotid IMT incrassation displayed enhanced MDA and protein carbonylation levels (P < 0.01), accompanied by activation and up‐regulation of NOX in monocytes (P < 0.05) compared with the controls. The monocytes isolated from five healthy donors were used for in vitro experiments. Reactive oxygen species (ROS) production and NOX expression in monocytes were examined. The results also indicated that CRP could promote the adhesion of monocyte‐endothelial cell by up‐regulation of MCP‐1 expression (P < 0.05). Importantly, NFκ B and p38 MAPK signaling pathways, which were activated by NOX‐derived ROS, were involved in CRP‐induced monocyte‐endothelial cell adhesion and up‐regulation of MCP‐1 expression. These data suggested that CRP could promote the adhesion of monocytes to endothelial cells via NOX‐mediated oxidative stress. J. Cell. Biochem. 113: 857–867, 2012. © 2011 Wiley Periodicals, Inc.     

Characterization and validation of a chemiluminescent assay based on 1,2-dioxetanes for rapid detection of viable Escherichia coli

[(4-methoxy-4(3-β-d-galactose-4-chlorophenyl)]spiro[1,2-dioxetane-3-1,3-tricyclo[7.3.1.0^2,7]tridec-2,7-ene] (“sβ-Gal 102”) and sodium [4-methoxy-4(3-β-d-glucuronic acid-4-chlorophenyl)]spiro[1,2-dioxetane-3-1,3-tricyclo[7.3.1.0^2,7]tridec-2,7-ene] (“sβ-Glucor 102”) are carbohydrate-containing 1,2-dioxetane compounds that produce chemiluminescence upon enzymatic hydrolysis by β-d-galactosidase, and β-d-glucuronidase, respectively. In this study, we have characterized and validated a sensitive detection principle for viable Escherichia coli based on enzymatic cleavage of sβ-Gal 102 and sβ-Glucor 102 (“ColiLight II”). The proposed chemiluminescent assay was optimized with respect to analytical requirements including incubation time, temperature, pH, enzyme induction, and cell permeabilization. The sensitivity and specificity rates of the assay were tested on ten different bacterial genera. The assay was found to be representative based on low coefficients of variations for both accuracy and precision. The analysis time was less than 1 h and the analytical detection limit was 10² to 10³ E. coli cells. In combination with membrane filtration and a brief resuscitation step of 4 h, the proposed assay was capable of detecting low concentrations of stressed E. coli in potable water (<30 CFU 100 ml⁻¹). The proposed chemiluminescent enzyme assay may be used for assessing the metabolic activity of E. coli in oligotrophic environments and for early warning detection of low concentrations of E. coli in water for human consumption.     

Hypertonic Saline Attenuates Colonic Tumor Cell Metastatic Potential by Activating Transmembrane Sodium Conductance

Hypertonic saline (HTS) suppresses tumor cell-endothelial interactions by reducing integrin expression. This translates into reduced adhesion, migration and metastatic potential. This study determined the relative contributions of hyperosmolarity and sodium-specific hypertonicity on the inhibitory effects of HTS, the intracellular pH and sodium responses to HTS and the role of cytoskeletal remodeling in these changes. Human colonic tumor cells (LS174T) were exposed to lipopolysaccharide under isotonic, hypertonic, sodium-free (N-methyl- D-glucamine), hyperosmolar (mannitol or urea), disrupted cytoskeletal (10 μg/ml cytochalasin D) conditions or in the presence of 5-(N-ethyl-N-isopropyl)amiloride (EIPA). β₁ integrin expression was measured flow-cytometrically. Intracellular sodium and pH were measured with confocal laser microscopic imaging. Statistical analysis was performed with analysis of variance, and P < 0.05 was considered significant. Data are represented as mean ± SEM. Hypertonic exposure attenuated integrin expression (62.03 ± 4.7% of control, P < 0.04). No discernible effect was observed with sodium-free or hyperosmolar solutions. HTS evoked a cellular alkalinization (by a mean 0.2 pH units) and an increase in cytosolic sodium concentration (by a mean 12.4 mM, P < 0.001) via upregulation of sodium-hydrogen exchange. Disassembly of actin microfilaments by cytochalasin D and antiporter inhibition with EIPA abrogated the effect of hypertonicity on integrin expression and intracellular sodium and pH (P < 0.05). HTS downregulates adhesion molecule expression via a hypertonic, sodium-specific, cytoskeletally mediated mechanism that involves activation of sodium-hydrogen exchange with associated changes in intracellular pH and sodium concentrations.     

Plant Growth Inhibitory Compounds from Aqueous Leachate of Wheat Straw

When seedlings of lettuce, cress, rice and wheat were incubated with the leachate of wheat straw, the roots growth of lettuce and garden cress were particularly inhibited. The leachate of wheat straw (100 g eq./l) showed 80.5 and 79.4% inhibition for lettuce and cress roots, respectively. The inhibitory activity was stronger as the concentration of wheat straw leachate was greater. This result indicates that allelochemical(s) inhibiting the roots growth of lettuce and cress are leached from the wheat straw into the water. Two potent compounds were isolated from the leachate of the wheat straw and identified as syringoylglycerol 9-O-β-d-glucopyranoside and l-tryptophan by spectral analyses. Syringoylglycerol 9-O-β-d-glucopyranoside inhibited the roots growth of lettuce and cress at concentrations greater than 0.1 and 10.0 μM, respectively. On the other hand, l-tryptophan inhibited the roots growth of lettuce and cress at concentrations greater than 0.1 and 1.0 μM, respectively. The content of syringoylglycerol 9-O-β-d-glucopyranoside and l-tryptophan in the leachate of wheat straw (100 g eq./l) was 18.4 ± 0.7 and 6.2 ± 0.6 μM, respectively. Syringoylglycerol 9-O-β-d-glucopyranoside (18.4 μM) showed 21.5 and 13.5% inhibition in the lettuce and cress roots assay, respectively. On the other hand, 6.2 μM of l-tryptophan showed 47.5 and 35.0% inhibition in the lettuce and cress roots assay, respectively. These results suggested that l-tryptophan may be a major contributor to the allelopathy in aqueous leachate of wheat straw and syringoylglycerol 9-O-β-d-glucopyranoside may be a minor contributor.     

Characterization of Na+-Coupled Glutamate/Aspartate Transport by a Rat Brain Astrocyte Line Expressing GLAST and EAAC1

d-Aspartate (d-Asp) uptake by suspensions of cerebral rat brain astrocytes (RBA) maintained in long-term culture was studied as a means of characterizing function and regulation of Glutamate/Aspartate (Glu/Asp) transporter isoforms in the cells. d-Asp influx is Na⁺-dependent with K _m = 5 μm and V _max= 0.7 nmoles · min⁻¹· mg protein⁻¹. Influx is sigmoidal as f[Na⁺] with _Na+ K _m ∼ 12 μm and Hill coefficient of 1.9. The cells establish steady-state d-Asp gradients >3,000-fold. Phorbol ester (PMA) enhances uptake, and gradients near 6,000-fold are achieved due to a 2-fold increase in V _max, with no change in K _m . At initial [d-Asp] = 10 μm, RBA take up more than 90% of total d-Asp, and extracellular levels are reduced to levels below 1 μm. Ionophores that dissipate the Δμ_Na+ inhibit gradient formation. Genistein (GEN, 100 μm), a PTK inhibitor, causes a 40% decrease in d-Asp. Inactive analogs of PMA (4α-PMA) and GEN (daidzein) have no detectable effect, although the stimulatory PMA response still occurs when GEN is present. Further specificity of action is indicated by the fact that PMA has no effect on Na⁺-coupled ALA uptake, but GEN is stimulatory. d-Asp uptake is strongly inhibited by serine-O-sulfate (S-O-S), threohydroxy-aspartate (THA), l-Asp, and l-Glu, but not by d-Glu, kainic acid (KA), or dihydrokainate (DHK), an inhibition pattern characteristic of GLAST and EAAC1 transporter isoforms. mRNA for both isoforms was detected by RT-PCR, and Western blotting with appropriate antibodies shows that both proteins are expressed in these cells. Received: 11 January 2001/Revised: 26 March 2001     

Biotechnological production of <Emphasis Type="SmallCaps">l</Emphasis>-ribose from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g⁻¹ h⁻¹ (1.84 ± 0.03 g l⁻¹ h⁻¹) and 0.27 ± 0.01 g g⁻¹ h⁻¹ (1.91 ± 0.1 g l⁻¹ h⁻¹) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose.     

A new route to l-threo-3-[4-(methylthio)phenylserine], a key intermediate for the synthesis of antibiotics: recombinant low-specificity d-threonine aldolase-catalyzed stereospecific resolution

A new enzymatic resolution process was established for the production of l-threo-3-[4-(methylthio)phenylserine] (MTPS), an intermediate for synthesis of antibiotics, florfenicol and thiamphenicol, using the recombinant low-specificity d-threonine aldolase from Arthrobacter sp. DK-38. Chemically synthesized dl-threo-MTPS was efficiently resolved with either the purified enzyme or the intact recombinant Escherichiacoli cells overproducing the enzyme. Under the optimized experimental conditions, 100 mM (22.8 g l⁻¹) l-threo-MTPS was obtained from 200 mM (45.5 g l⁻¹) dl-threo-MTPS, with a molar yield of 50% and a 99.6% enantiomeric excess. Received: 2 September 1998 / Received revision: 27 October 1998 / Accepted: 29 November 1998     

<Emphasis Type="Italic">QUASIMODO1</Emphasis> is expressed in vascular tissue of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> inflorescence stems,and affects homogalacturonan and xylan biosynthesis

An insertion in the promoter of the Arabidopsis thaliana QUA1 gene (qua1-1 allele) leads to a dwarf plant phenotype and a reduction in cell adhesion, particularly between epidermal cells in seedlings and young leaves. This coincides with a reduction in the level of homogalacturonan epitopes and the amount of GalA in isolated cell walls (Bouton et al., Plant Cell 14: 2577 2002). The present study was undertaken in order to investigate further the link between QUA1 and cell wall biosynthesis. We have used rapidly elongating inflorescence stems to compare cell wall biosynthesis in wild type and qua1-1 mutant tissue. Relative to the wild type, homogalacturonan α-1-4-D-galacturonosyltransferase activity was consistently reduced in qua1-1 stems (by about 23% in microsomal and 33% in detergent-solubilized membrane preparations). Activities of β-1-4-D-xylan synthase, β-1-4-D-galactan synthase and β-glucan synthase II activities were also measured in microsomal membranes. Of these, only β-1-4-D-xylan synthase was affected, and was reduced by about 40% in qua1-1 stems relative to wild type. The mutant phenotype was apparent in inflorescence stems, and was investigated in detail using microscopy and cell wall composition analyses. Using in situ PCR techniques, QUA1 mRNA was localized to discrete cells of the vascular tissue and subepidermal layers. In mutant stems, the organization of these tissues was disrupted and there was a modest reduction in homogalacturonan (JIM5) epitopes. This study demonstrates a specific role for QUA1 in the development of vascular tissue in rapidly elongating inflorescence stems and supports a role of QUA1 in pectin and hemicellulose cell wall synthesis through affects on α-1,4-D-galacturonosyltransferase and β-1,4-D-xylan synthase activities.     

Macro elements in inoculation and co-cultivation medium strongly affect the efficiency of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Lilium</Emphasis>

A highly efficient Agrobacterium-mediated transformation system for Lilium × formolongi was established by modifying the medium used for inoculation and co-cultivation. Meristematic nodular calli of Lilium were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm harboring an intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase, and neomycin phosphotransferase II genes. The effects of ten different types of media and carbohydrates (sucrose, d-glucose, and l-arabinose) in both inoculation and co-cultivation media were evaluated. Interestingly, a dramatic increase in the frequency of transformation (25.4%) was observed when Murashige and Skoog (MS) medium containing sucrose and lacking KH₂PO₄, NH₄NO₃, KNO₃, and CaCl₂ was used. Hygromycin-resistant transgenic calli were obtained only in medium supplemented with sucrose. The effects of this modified medium were also investigated for Lilium cultivars ‘Acapulco’, ‘Casa Blanca’, and ‘Red Ruby’. The highest frequency of transformation (23.3%) was obtained for cv. Acapulco. Hygromycin-resistant calli were successfully regenerated into plantlets on plant growth regulator-free MS medium. Transgenic plants were confirmed by GUS histochemical assay, polymerase chain reaction (PCR), and Southern blot analyses.     

Antimicrobial activity of various cationic molecules on foodborne pathogens

Antibacterial effects of various arginine- and lysine-rich polycationic proteins and polymers were evaluated by broth and solid dilution assay on a range of foodborne pathogens, Gram-positive and Gram-negative bacteria. The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of α-poly-l-lysine (poly-lys), α-poly-l-arginine (poly-arg) and protamines from herring sperm (clupeine sulphate) and salmon sperm (salmine sulphate) were determined on Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Shigella sonnei, Escherichia coli O157:H7 and Pseudomonas aeruginosa. All these molecules showed antibacterial activity on all strains with different MIC and MBC values. The molecular mechanisms underlying the effect of α-poly-l-arginine might be related to the entrance of the molecule into the cell. In fact α-poly-l-arginine labelled with 7-Diethylamino coumarin-3-carboxylic acid, succinimidyl ester (DEAC,SE) showed ability to permeate the cell membrane of B. cereus and E. coli O157:H7.     

Expression in Escherichia coli and characterization of β-xylosidases GH39 and GH-43 from Bacillus halodurans C-125

To develop xylosidases as tools for the hydrolysis of wheat bran arabinoxylans, two β-xylosidases from Bacillus halodurans C-125 have been cloned and expressed in Escherichia coli. The recombinant (His)₆-tagged enzymes, designated as XylBH39 and XylBH43, were efficiently purified using Ni²⁺-affinity chromatography. Determination of native molecular masses indicated that XylBH43 is dimeric in solution, whereas a similar analysis of XylBH39 did not allow differentiation between the dimeric and trimeric states. Both enzymes had similar pH and temperature optima (pH 7.5 and 55 °C for XylBH39 and pH 8 and 60 °C for XylBH43) and were relatively stable over the pH range of 3.5–8.5. In contrast, XylBH39 was more thermostable. At 60 °C, XylBH39 and XylBH43 displayed approximate half-life values of 2.40 and 0.05 h, respectively. The comparison of the ratio k _cat/K _M revealed that XylBH43 hydrolyzed p-nitrophenyl-β-d-xyloside more efficiently (4.6-fold) than XylBH39. Similarly, while XylBH43 was 18-fold less active on p-nitrophenyl-α-l-arabinofuranoside, XylBH39 was essentially inactive on this substrate. Using either p-nitrophenyl-β-d-xyloside or xylotriose, XylBH39 performed transglycosylation, while xylobiose proved to be a poor substrate for both hydrolysis and transglycosylation. The use of XylBH39 and XylBH43 for the posttreatment of endoxylanase-generated wheat bran hydrolysates revealed that XylBH43 efficiently produced xylose monomers (385 μg/ml after 330 min incubation). Its activity was improved by the simultaneous deployment of an α-l-arabinofuranosidase. Together, these enzymes were able to release 521 μg/ml of xylose after 330 min. This constitutes an approximate yield improvement of 35%.     

Age and composition of carbonate shoreface sediments, Kailua Bay, Oahu, Hawaii

The origin, age, and dynamics of carbonate sediments in Kailua Bay on Oahu, Hawaii, are described. The shoreface (from shoreline to 4 km offshore) consists of a broad (5 km²) fringing coral reef ecosystem bisected by a sinuous, shore-normal, sand-filled paleostream channel 200–300 m wide. The median grain diameter of surface sands is finest on the beach face (<0.3 mm) and increases offshore along the channel axis. Kailua sands are >90% biogenic carbonate, dominated by skeletal fragments of coralline algae (e.g. Porolithon, up to 50%) followed by the calcareous green alga Halimeda (up to 32%), coral fragments (1–24%), mollusc fragments (6–21%), and benthic foraminifera (1–10%). Sand composition and age across the shoreface are correlated to carbonate production. Corals and coralline algae, principal builders of the reef framework, are younger and more abundant in sands along the channel axis and in offshore reefal areas, while Halimeda, molluscs, and foraminifera are younger and more dominant in nearshore waters shoreward of the main region of framework building. Shoreface sediments are relatively old. Of 20 calibrated radiocarbon dates on skeletal constituents of sand, only three are younger than 500 years b.p.; six are 500–1000 years b.p.; six are 1000–2000 years b.p.; and five are 2000–5000 years b.p. Dated fine sands are older than medium to coarse sands and hence may constitute a reservoir of fossil carbonate that is distributed over the entire shoreface. Dominance of fossiliferous sand indicates long storage times for carbonate grains, which tend to decrease in size with age, such that the entire period of relative sea-level inundation (∼5000 years) is represented in the sediment. Despite an apparently healthy modern coral ecosystem, the surficial sand pool of Kailua Bay is dominated by sand reflecting an antecedent system, possibly one that existed under a +1–2 m sea-level high stand during the mid- to late Holocene. Accepted: 20 December 1999     

Cloning,sequencing, overexpression and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-rhamnose isomerase from <Emphasis Type="Italic">Bacillus pallidus</Emphasis> Y25 for rare sugar production

The l-rhamnose isomerase gene (L -rhi) encoding for l-rhamnose isomerase (l-RhI) from Bacillus pallidus Y25, a facultative thermophilic bacterium, was cloned and overexpressed in Escherichia coli with a cooperation of the 6×His sequence at a C-terminal of the protein. The open reading frame of L -rhi consisted of 1,236 nucleotides encoding 412 amino acid residues with a calculated molecular mass of 47,636 Da, showing a good agreement with the native enzyme. Mass-produced l-RhI was achieved in a large quantity (470 mg/l broth) as a soluble protein. The recombinant enzyme was purified to homogeneity by a single step purification using a Ni-NTA affinity column chromatography. The purified recombinant l-RhI exhibited maximum activity at 65°C (pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be retained after incubation at 60°C for 60 min. The apparent affinity (K _m) and catalytic efficiency (k _cat/K _m) for l-rhamnose (at 65°C) were 4.89 mM and 8.36 × 10⁵ M⁻¹ min⁻¹, respectively. The enzyme demonstrated relatively low levels of amino acid sequence similarity (42 and 12%), higher thermostability, and different substrate specificity to those of E. coli and Pseudomonas stutzeri, respectively. The enzyme has a good catalyzing activity at 50°C, for d-allose, l-mannose, d-ribulose, and l-talose from d-psicose, l-fructose, d-ribose and l-tagatose with a conversion yield of 35, 25, 16 and 10%, respectively, without a contamination of by-products. These findings indicated that the recombinant l-RhI from B. pallidus is appropriate for use as a new source of rare sugar producing enzyme on a mass scale production.     

Vacuolar Chloride Transport in Mesembryanthemum crystallinum L. Measured Using the Fluorescent Dye Lucigenin

To study vacuolar chloride (Cl⁻) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl⁻ uptake into isolated tonoplast vesicles was measured using the Cl⁻-sensitive fluorescent dye lucigenin (N,N′-dimethyl-9,9′-bisacridinium dinitrate). Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm, respectively, and showed a high sensitivity towards Cl⁻, with a Stern-Volmer constant of 173 m ⁻¹ in standard assay buffer. While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched, if at all, by other anions. However, the fluorescence intensity and Cl⁻-sensitivity of lucigenin was shown to be strongly affected by alkaline pH and was dependent on the conjugate base used as the buffering ion. Chloride transport into tonoplast vesicles of M. crystallinum loaded with 10 mm lucigenin showed saturation-type kinetics with an apparent K _m of 17.2 mm and a V _max of 4.8 mm min⁻¹. Vacuolar Cl⁻ transport was not affected by sulfate, malate, or nitrate. In the presence of 250 μm p-chloromercuribenzene sulfonate, a known anion-transport inhibitor, vacuolar Cl⁻ transport was actually significantly increased by 24%. To determine absolute fluxes of Cl⁻ using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be 1.13 × 10⁷ m⁻¹. After correcting for a 4.4-fold lower apparent Stern-Volmer constant for intravesicular lucigenin, a maximum rate of Cl⁻ transport of 31 nmol m⁻² sec⁻¹ was calculated, in good agreement with values obtained for the plant vacuolar membrane using other techniques. Received: 18 February 2000/Revised: 30 June 2000