Preparation of a glycosynthase from the β-glycosidase of the Archaeon Pyrococcus horikoshii

The β-glycosidase from the hyperthermophilic Archaeon Pyrococcus horikoshii (Phoβ-gly) is a monomeric enzyme with wide substrate specificity belonging to family 1 of glycoside hydrolases classification. Inspection of the three-dimensional structure of the enzyme, recently resolved, showed that Phoβ-gly is membrane bound and that the residues putatively involved in the catalytic activity are Glu155 and Glu324 working as the general acid/base and the nucleophile of the reaction, respectively. We show here that mutation of the latter completely eliminated the activity of the enzyme and that it could be reactivated in the presence of sodium formate. Analysis of the products obtained in the presence of sodium formate buffer pH 4.0 at 75°C showed that the Glu324Gly mutant acts as a hyperthermophilic glycosynthase.     

Preparation of Aryl β-d-Mannopyranoside Derivatives via β-d-arabino-Hexopyranosiduloses

Sequential oxidation and reduction of aryl 4, 6-O-benzylidene-β-d-glucosides with dimethyl sulfoxide-phosphorus pentoxide mixture (DMSO–P₂O₅) and sodium borohydride were carried out as a new means for the preparation of aryl β-d-mannopyranoside derivatives. p-Nitrophenyl 4, 6-O-benzylidene-β-d-mannopyranoside was obtained in 22% yield from the corresponding glucoside 3-O-acetate, whereas from the unprotected acetal, 4, 6-O-benzylidene acetals of the corresponding mannoside and alloside were isolated in the yields of 6.7 and 2.1%, respectively. Similarly, phenyl 4, 6-O-benzylidene β-d-mannoside, alloside, and altroside were obtained from the corresponding glucoside in 2.2, 0.8 and 2.1% yields, respectively.     

Preparation and characterization of biotinylated probes for the β-interferon receptor

• 1.1. Recently we described the isolation of the β-interferon receptor [Zhang et al. (1986) J. biol. Chem. 261, 8017–8021]. A highly purified product was obtained but in low quantities.
• 2.2. The use ofbiotinylated β-interferon as a ligand represents an alternate approach to receptor isolation.
• 3.3. We have prepared and characterized the derivatives N-(biotinyl)- and N-(biotinyl-ϵ-aminocaproyl)-recombinant human [Ser¹⁷-interferon β (B- and BC-recHulFNβ).
• 4.4. Biotin incorporation does not result in any loss of antiviral activity, demonstrating the recognition of the derivative by the cell receptor.
• 5.5. The biotinylated recHuIFNβ binds specifically and reversibly to succinoylavidin or guanidine thiocyanate-stripped succinoylavidin linked to a Sepharose matrix.
• 6.6. Comparison of the competition curves obtained with [¹⁴C]biotin and [³H]biotinyl recHuIFN, in the presence of increasing concentrations of biotin suggests that the IFN moiety of the derivative has little effect on the affinity of biotin for avidin.
• 7.7. Biotinylated recHuIFNβ derivatives represent useful probes for the β-IFN receptor.

     

Evidence for the participation of calmodulin in stimulus–secretion coupling in the pancreatic β-cell

1. The ability of a range of phenothiazines to inhibit activation of brain phosphodiesterase by purified calmodulin was studied. Trifluoperazine, prochlorperazine and 8-hydroxyprochlorperazine produced equipotent dose-dependent inhibition with half-maximum inhibition at 12mum. When tested at 10 or 50mum, 7-hydroxyprochlorperazine was a similarly potent inhibitor. However, trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were ineffective at concentrations up to 50mum, and produced only a modest inhibition at 100mum. 2. The same phenothiazines were tested for their ability to inhibit activation of brain phosphodiesterase by boiled extracts of rat islets of Langerhans. At a concentration of 20mum, 70-80% inhibition was observed with trifluoperazine, prochlorperazine, 7-hydroxyprochlorperazine or 8-hydroxyprochlorperazine, whereas trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were less effective. 3. The effect of these phenothiazines on insulin release from pancreatic islets was studied in batch-type incubations. Insulin release stimulated by glucose (20mm) was markedly inhibited by 10mum-trifluoperazine or -prochlorperazine and further inhibited at a concentration of 20mum. 8-Hydroxyprochlorperazine (20mum) was also a potent inhibitor but 7-hydroxyprochlorperazine (20mum) elicited only a modest inhibition of glucose-stimulated insulin release; no inhibition was observed with trifluoperazine-5-oxide or N-methyl-2-(trifluoromethyl)phenothiazine. 4. Trifluoperazine (20mum) markedly inhibited insulin release stimulated by leucine or 4-methyl-2-oxopentanoate in the absence of glucose, and both trifluoperazine and prochlorperazine (20mum) decreased insulin release stimulated by glibenclamide in the presence of 3.3mm-glucose. 5. None of the phenothiazines affected basal insulin release in the presence of 2mm-glucose. 6. Trifluoperazine (20mum) did not inhibit islet glucose utilization nor the incorporation of [(3)H]leucine into (pro)insulin or total islet protein. 7. Islet extracts catalysed the incorporation of (32)P from [gamma-(32)P]ATP into endogenous protein substrates. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis resolved several phosphorylated bands, but incorporation was slight. However, calmodulin in the presence of Ca(2+) greatly enhanced incorporation: the predominant phosphorylated band had an estimated mol.wt. of 55000. This enhanced incorporation was abolished by trifluoperazine, but not by cyclic AMP-dependent protein kinase inhibitor protein. 8. These results suggest that islet phosphodiesterase-stimulating activity is similar to, although not necessarily identical with, calmodulin from skeletal muscle; that islet calmodulin may play an important role in Ca(2+)-dependent stimulus-secretion coupling in the beta-cell; and that calmodulin may exert part at least of its effect on secretion via phosphorylation of endogenous islet proteins.     

The arsenolysis reaction in the biotechnological method of synthesis of modified purine β-D-arabinonucleosides

We found a unique property of E. coli purine nucleoside phosphorylases to selectively perform the arsenolysis reaction of ribonucleosides in their active site without affecting β-D-arabinonucleosides. In the synthesis of modified β-D-arabinonucleosides from the corresponding ribonucleosides, the catalytical amount of sodium arsenate in the transglycosylation reaction provided a 95 to 98% conversion rate. Such an approach was shown to simplify the composition of the reaction mixtures and facilitate the isolation of the target nucleosides, particularly, vidarabine, fludarabine, and nelarabine.     

Preparation and immunological characterization of β-lactoglobulin-amylose conjugate

β-Lactoglobulin (BLG), a major allergen of cow's milk, was conjugated with the N-hydroxysuccinimide ester of the amylose-glycylglycine adduct (AG-ONSu) to reduce its immunogenicity, and the biochemical and immunological properties of the resulting conjugate (AG-BLG) were studied. The conjugate was prepared by modifying BLG with AG-ONSu, and was purified in a Sephadex G-100 column. The analytical data for AG-BLG indicated that 10.5 moles of AG-ONSu, with a mean molecular weight of 2,800, was covalently attached to the amino groups of the BLG molecule. Conjugation with AG-ONSu greatly decreased the reactivity of BLG with anti-BLG polyclonal antibodies owing to its shielding action for epitopes on the protein's surface. These findings suggest that AG-ONSu can be used advantageously to suppress the hypersensitivity mediated by IgG antibodies in milk allergy.     

Phosphorylation of β-lactoglobulin using amino acids as the sole base and nucleophile of the reaction

β-Lactoglobulin was phosphorylated with 20, 40, and 80 mol of POCl₃/mol protein in the presence of 4, 5, and 6 molar excess of basic amino acid per mol POCl₃. Maximal phosphorylation yields of 5 and 3 mol P/mol protein were achieved when the highest stoichiometries of POCl₃/arginine and lysine were used. Proportional high amounts of basic amino acids were also grafted to the protein molecule during its phosphorylation through the phosphoamide bond. Modified proteins displayed increased negative charges and reduced isoelectric points and were monomeric. The phosphorylated and phosphoamidatedβ-lactoglobulin showed improved functional properties.     

Molecular basis of β-thalassemia in Thailand: analysis of β-thalassemia mutations using the polymerase chain reaction

Summary -Thalassemia mutations in 71 chromosomes of Thai patients from the northeast, the middle and the south of the country were investigated using dot blot hybridization of PCR (polymerase chain reaction)-amplified DNA with allelespecific oligonucleotide probes. Eight different known molecular defects were detected, at different frequencies. There was an amber mutation in codon 17, a C-T transversion at position 654 of IVS-2, a frameshift mutation between codons 71 and 72, an A-G transition at nucleotide -28 within the TATA box (known as Chinese mutations), a G-T transversion at position 1 of IVS-1 (an Indian mutation), a 4bp deletion in codons 41/42 and a G-C transversion at position 5 of IVS-1 (described as both Chinese and Indian mutations) and a Thai original mutation, an ochre mutation in codon 35. Analysis of the three unknown alleles by DNA sequencing of the cloned DNA fragment amplified by PCR revealed an A-G substitution at the second position of the codon for amino acid 19 (AAC-AGC). The analytic approach used in the present study and the characteristic distribution of mutations in each region of Thailand will prove useful for setting up a prenatal diagnosis program.     

Recognition of β-hairpin motifs in proteins by using the composite vector

A composite vector method for predicting β-hairpin motifs in proteins is proposed by combining the score of matrix, increment of diversity, the value of distance and auto-correlation information to express the information of sequence. The prediction is based on analysis of data from 3,088 non-homologous protein chains including 6,035 β-hairpin motifs and 2,738 non-β-hairpin motifs. The overall accuracy of prediction and Matthew’s correlation coefficient are 83.1% and 0.59, respectively. In addition, by using the same methods, the accuracy of 80.7% and Matthew’s correlation coefficient of 0.61 are obtained for other dataset with 2,878 non-homologous protein chains, which contains 4,884 β-hairpin motifs and 4,310 non-β-hairpin motifs. Better results are also obtained in the prediction of the β-hairpin motifs of proteins by analysis of the CASP6 dataset.     

Galactosylation of antibiotics using the β-galactosidase from Aspergillus oryzae

The β-galactosidase from Aspergillus oryzae has been shown to catalyze the synthesis of β-galactosides of antibiotics such as chlorphenisin and chloramphenicol using β-lactose as the galactosyl donor. Among the water-miscible organic solvents tested, 20% (v/v) acetonitrile in the reaction mixture gave the highest yield in galactoside synthesis. The products obtained were purified by preparative TLC and liquid chromatography and analyzed by ¹H-and ¹³C-NMR, and MS (FAB). Chlorphenisin and chloramphenicol were galactosylated exclusively at their primary hydroxy groups. The pH optimum for the transgalactosylation reaction was between pH 4–5. Increasing concentrations of galactosyl donor and aglycon caused increasing yields of galactosides. When the resulting galactosylated antibiotic was withdrawn from the sample, further synthesis was observed. This could be accelerated either by withdrawing the resulting monosaccharides (glucose and galactose) or exchanging them for mannose.     

A simple method using β-globin polymerase chain reaction for the species identification of animal cell lines—A progress report

Continuous cell lines are widely used in cell biology and serve as model systems in basic and applied research. Fundamental requirements for the use of cell lines are a well-identified origin and the exclusion of cross-contamination by prokaryotic or eukaryotic cells. Because the cross-contamination of one cell line with another cell line may occur in a concealed manner, special emphasis must be taken to (1) prevent such an "accident" and (2) monitor regularly the identity of the cell line(s) in use. Apart from human cell lines, mouse-, rat-, and hamster-derived cell lines are used in basic cell culture and biotechnology. We established a polymerase chain reaction (PCR) assay to detect and confirm the species origin for these species and to detect interspecies cross-contamination. Our PCR method is based on oligonucleotide primers annealing to specific sequences in the beta-globin gene, which were designed to amplify one deoxyribonucleic acid (DNA) segment only per analyzed sample. We confirmed the species identity of 82 cell lines as human, mouse, rat, and Syrian hamster by beta-globin PCR. The DNAs from eight additional cell lines of less frequently used species were not amplified with the primers chosen. Cross-contamination of 5-10% of either mouse or rat DNA was detectable. One species-specific primer pair was sufficient for confirmation of the expected species, and for identification of an unknown cell line the combination of two or more primer pairs is suggested. Our PCR assay represents a powerful, fast, easy, robust, and inexpensive method for speciation and does not need any elaborate sequencing or computer-based analysis system.     

Novel method for aroma recovery from the bioconversion of lutein to β-ionone by Trichosporon asahii using a mesoporous silicate material

In this work, we report on the synthesis and ability of the mesoporous material MCM-41 to adsorb the norisoprenoid β-ionone. This compound, with a violet aroma note, can be produced from lutein by the yeast Trichosporon asahii through a bioconversion process. We found that β-ionone inhibited the yeast growth and constrained aroma formation. Growth inhibition was overcome using silicate MCM-41 as sorbent device in a fermentation system that allowed product removal from the culture medium by headspace manipulation. Compared to a commercial silica gel, the mesoporous material exhibited a 4.5-fold higher β-ionone adsorption. Contrasting to cultures without the sorbent device, the presence of MCM-41 allowed a marked increase (14-fold) in β-ionone production. Our results suggested that confinement of the norisoprenoid into the sorbent material bypassed its toxicity which allowed a better β-ionone production. This study represents the first report on the use of MCM-41 to recover an aroma produced by fermentation and therefore, a novel application for a mesoporous material.     

Effect of transmembrane Ca2+ gradient on the coupling of β-adrenergic receptors and adenylyl cyclase

In order to investigate the effect of transmembrane Ca²⁺ gradient on G_s mediated coupling of -AR and adenylyl cyclase, -AR from duck erythrocytes and G_s and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca²⁺ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca^2– gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated -AR via coupling to G_s and can be further enhanced by the decrease of such Ca²⁺ gradient within certain range (100 fold) following Ca²⁺ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca²⁺ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of -AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled -AR in those proteoliposomes. It is suggested that a proper transmembrane Ca²⁺ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted -AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated -AR via Gs.Abbreviations ATP adenosine triphosphate - -AR -adrenergic receptors - AC adenylyl cyclase - DHA dihydroalprenolol - DPH diphenylhexatriene - [Ca²⁺]_i Ca²⁺ concentration inside proteoliposomes - [Ca²⁺]_o Ca²⁺ concentration outside proteoliposomes - cAMP cyclic adenosine monophosphate - DTT Dithiothreitol - FS fluorescein sulfonate - G_s Stimulatory GTP-binding protein - GTP guanosine triphosphate - GTPS guanosine 5-O-(3-thiotriphosphate) - kDa kilodalton - SDS sodium dodecyl sulfate - Tris N-tris(hydroxymethyl)aminomethane     

Characterization of the G protein coupling of SRIF and β-adrenergic receptors to the maxi KCa channel in insulin-secreting cells

Modulation of the Ca- and voltage-dependent K channel—K_Ca—by receptors coupled to the G proteins G _i /G _o and G _s has been studied in insulin-secreting cells using the patch clamp technique. In excised outside-out patches somatostatin (somatotropin-releasing inhibitory factor; SRIF) caused concentration-dependent inhibition of the K_Ca channel, an effect that was prevented by pertussis toxin (PTX). In inside-out patches, exogenous subunits of either G _i or G _o -type G proteins also inhibited the K_Ca channel (IC₅₀ 5.9 and 5.7 pM, respectively). These data indicate that SRIF suppresses K_Ca channel activity via a membrane-delimited pathway that involves the subunits of PTX-sensitive G proteins G _i and/or G _o . In outside-out patches, activation of G _s either by -agonists or with cholera toxin (CTX) increased K_Ca channel activity, consistent with a membrane-delimited stimulatory pathway linking the -adrenergic receptor to the K_Ca channel via G _s . In outside-out patches, channel inhibition by SRIF suppressed the stimulatory effect of -agonists but not that of CTX, while in inside-out patches CTX reversed channel inhibition induced by exogenous _i or _o . Taken together these data suggest that K_Ca channel activity is enhanced by activation of G _s and blocked by activated G _i and/or G _o . Further, K_Ca channel stimulation by activated G _s may be direct, while inhibition by G _i /G _o may involve deactivation of G _s . In inside-out patches K_Ca channel activity was reduced by an activator of protein kinase C (PKC) and enhanced by inhibitors of PKC, indicating that PKC also acts to inhibit the K_Ca channel via a membrane delimited pathway. In outside-out patches, chelerythrine, a membrane permeant inhibitor of PKC prevented the inhibitory effect of SRIF, and in inside-out patches PKC inhibitors prevented the inhibitory effect of exogenous _i or _o . These data indicate that PKC facilitates the inhibitory effect of the PTX-sensitive G proteins which are activated by coupling to SRIF receptors. To account for these results a mechanism is proposed whereby PKC may be involved in G _i /G _o -induced deactivation of G _s .The authors would like to thank Dr. S. Ciani for many helpful discussions, Dr. A.E. Boyd III for supplying the HIT cells, Drs. J. Codina and L. Birnbaumer for supplying the alpha subunits of the G proteins G _i and G _o , and Mrs. Satoko Hagiwara for preparing and maintaining the cell cultures.This work was supported by grant DCB-8919368 from the National Science Foundation and a research grant (W-P 880513) from the American Diabetes Association to B.R., and by grant RO1-DK39652 from the National Institutes of Health to G.T.E.     

Calculation of binding affinities of HIV-1 RT and β-secretase inhibitors using the linear interaction energy method with explicit and continuum solvation approaches

The linear interaction energy (LIE) approach has been applied to estimate the binding free energies of representative sets of HIV-1 RT and β-Secretase inhibitors, using both molecular dynamics (MD) and tethered energy minimization sampling protocols with the OPLS-AA potential, using a range of solvation methodologies. Generalized Born (GB), ‘shell’ and periodic boundary condition (PBC) solvation were used, the latter with reaction field (RF) electrostatics. Poisson-Boltzmann (PB) and GB continuum electrostatics schemes were applied to the simulation trajectories for each solvation type to estimate the electrostatic ligand-water interaction energy in both the free and bound states. Reasonable agreement of the LIE predictions was obtained with respect to experimental binding free energy estimates for both systems: for instance, ‘PB’ fits on MD trajectories carried out with PBC solvation and RF electrostatics led to models with standard errors of 1.11 and 1.03 kcal mol⁻¹ and coefficients of determination, r ² of 0.76 and 0.75 for the HIV-1 RT and β-Secretase sets. However, it was also found that results from MD sampling using PBC solvation provided only slightly better fits than from simulations using shell or Born solvation or tethered energy minimization sampling. Figure Evolution of the running averages for compound H11 (binding to HIV-1RT) of the bound state ligand-water and ligand-protein interaction energies. The ligand-water electrostatic terms are twice the corresponding GB and PB electrostatic solvation free energies. The ligand-receptor van der Waals and Coulombic interaction energies are also shown, in addition to the ligand-water van der Waals interaction term. The terms were calculated (without application of a cut-off) from a trajectory sampled under PBC solvation with reaction field electrostatics Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Preparation and Physicochemical Characterization of Amoxicillin β-cyclodextrin Complexes

Amoxicillin (AMOX), a penicillin A, belongs to the β-lactam family It is usually the drug of choice within the class because it is better absorbed, following oral administration, than other β-lactam antibiotics. Its β-lactamase degradation might be prevented by using a molecular [AMOX:β-CD] complex. The aim of this work was to prepare complexes using two methods and then characterize interactions between AMOX and the native β-CD. The extent of complexation in solution has been evaluated by high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and 2D rotating-frame Overhauser enhancement spectroscopy (2D ROESY). Mass changes (TG), calorimetric effects (DSC), and mass spectrometry (MS) were determined on the same sample under identical conditions using the Skimmer coupling system. Skimmer and infrared spectroscopy (FT-IR) were used to characterize the solid state of the binary system. Complexation of AMOX with β-CD was proven by FT-IR, NMR, DSC, and HPLC. The 2D ROESY spectra did not show any dipolar proton interaction of the AMOX with cyclodextrin. The 1:1 stoichiometry of the complex was obtained by HPLC. The stability constant for AMOX with β-CD was determined to be 1,878 M⁻¹. In the [AMOX:β-CD] complex, the phenyl group is included inside the β-CD, and the ionized carboxyl group on the penam ring forms hydrogen bonds with the secondary hydroxyl groups of another β-CD to keep the complex stable. Preparation methods allowed exactly the same complex.     

Enhanced β-fructofuranosidase biosynthesis by Rhodotorula glutinis using Taguchi robust design method

The aim of this study was to produce β-fructofuranosidase enzyme by Rhodotorula glutinis SO28, using sugar beet (Beta vulgaris) as carbon source due to its high sucrose content and easy availability. β-Fructofuranosidase production was carried out in submerged fermentation. Taguchi orthogonal array (OA) design of experiment (DOE) method was employed for optimization process of β-fructofuranosidase production by R. glutinis SO28. An OA layout of L16 was constructed with five influential factors on β-fructofuranosidase biosynthesis namely, carbon source (sugar beet), initial pH, incubation temperature, agitation speed and incubation time. The average results of β-fructofuranosidase yield obtained from the determined 16 batches were processed with Minitab^® 16.2.3 software at “larger is better” as quality character. The results showed that the maximum β-fructofuranosidase activity was obtained as 21.11?±?0.47?U/mL, which was close to the predicted result (21.78?±?0.43?U/mL). Consequently, sugar beet can be suggested as an economical substrate for β-fructofuranosidase production. Besides, use of Taguchi DOE enhanced enzyme activity about 3-fold when compared with unoptimized condition.     

Introducing site-specific glycosylation using protein engineering techniques reduces the immunogenicity of β-lactoglobulin

To reduce the immunogenicity of β-lactoglobulin (BLG), we prepared wild-type bovine BLG variant A (wt) and three site-specifically glycosylated BLGs (D28N, D137N/A139S, and P153A), and expressed them in the methylotrophic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the α-factor signal peptide from Saccharomyces cerevisiae. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the glycosylated BLGs were conjugated with a ~4 kDa high-mannose chain. Each glycosylated BLG retained ～80% of the retinol-binding activity of BLG. Structural analyses by intrinsic fluorescence, CD spectra, and ELISA with monoclonal antibodies indicated that the surface structure was slightly changed by using protein engineering techniques, but that the site-specifically glycosylated BLGs were covered by high-mannose chains without substantial disruption of wt conformation. Antibody responses to the glycosylated BLGs tended to be weaker in BALB/c, C57BL/6, and C3H/He mice. We conclude that site-specific glycosylation is an effective method to reduce the immunogenicity of BLG, and that masking of epitopes by high-mannose chains is effective to reduce immunogenicity.