Synthesis of alpha-lactosyl-(1-->3)-L-glycero-alpha-D-manno-heptopyranoside, a partial oligosaccharide structure expressed within the lipooligosaccharide produced by Neisseria gonorrhoeae strain 15253.

The glycosyl donor, hepta-O-benzyl-beta-lactosyl trichloroacetimidate (4) was prepared by treating hepta-O-benzyl-lactose with trichloroacetonitrile in the presence of potassium carbonate. The acceptor, methyl 2-O-benzyl-4,6-O-benzylidene-7,8-dideoxy-alpha-D-manno-oct-7-enopyranoside (8) was synthesized by hydrolysis of a 3,4-butane diacetal of methyl L-glycero-alpha-D-manno-oct-enopyranoside and subsequent benzylidenation. Glycosidation of the donor 4 with the acceptor 8 in 1,4-dioxane using Me(3)SiOTf as a promoter for 1 h at room temperature gave methyl (2,3,4,6-tetra-O-benzyl-beta-D-galactopyranosyl)-(1-->4)-(2,3,6-tri-O-benzyl-alpha-D-glucopyranosyl)-(1-->3)-2-O-benzyl-4,6-O-benzylidene-7,8-dideoxy-alpha-D-manno-oct-7-enopyranoside (9) as a major product (59%). The oct-enopyranoside moiety of the trisaccharide 9 was converted to a heptopyranoside (80%) by oxidative cleavage with OsO(4)-NaIO(4) and subsequent reduction. Hydrogenolysis of the resulting trisaccharide and subsequent acetylation gave the peracetate of alpha-lactosyl-(1-->3)-Hep. Deacetylation of the peracetate afforded the title trisaccharide.     

Nanometer-scale immobilization of polysaccharides on hydrophobic polymer plates in supercritical fluoroform/water emulsions

Hydrophilic polysaccharides such as dextran and hyaluronan were immobilized on a hydrophobic polystyrene (PSt) plate by a nanometer-scale surface penetration method in the emulsion of aqueous solutions in supercritical fluoroform (scCHF3). Since a supercritical fluid has high diffusiveness, water emulsions of polysaccharides can penetrate into the polymer surface. Dextran was surface-penetrated by two different methods: (1) the penetration of sucrose as a glucose donor and then the enzymatic polymerization to dextran near the surface catalyzed by dextransucrase, and (2) the direct penetration of dextran polymer into the PSt plate. The contact angle for water of the dextran-penetrated PSt plate was decreased to 78 degrees from 95 degrees of the untreated plate. The surface coverage and the penetration depth of polysaccharides could be obtained to be 10-30% and 10-20 nm, respectively, by X-ray photoelectron spectroscopy. These values could be controlled by the pressure of scCHF3. The transparency of the PSt dish did not change after the dextran penetration. Dextrans on the PSt plate could be elongated enzymatically by dextransucrase in the presence of sucrose as a glucose donor, and be detected by the enzyme-linked biotin-avidin assay. When anionic hyaluronan was surface-penetrated on the PSt plate instead of the neutral dextran, the plate showed the specific adhesion for human T-cells having hyaluronan receptors.     

Structural analysis of 2',3'-dideoxyinosine, 2',3'-dideoxyadenosine, 2',3'-dideoxyguanosine and 2',3'-dideoxycytidine by 500-MHz 1H-NMR spectroscopy and ab-initio molecular orbital calculations.

Solution structure of anti-AIDS drug, 2',3'-dideoxyinosine (ddI) has been assessed by NMR spectroscopy and pseudorotational analysis in conjunction with its analogues: 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxyguanosine (ddG) and 2',3'-dideoxycytidine (ddC). The absence of 3'-hydroxyl groups in these compounds has prompted us to establish the relationship between proton-proton and corresponding endocyclic torsion angles in the 2',3'-dideoxyribofuranose moiety on the basis of five available crystal structures of 2',3'-dideoxynucleosides. A subsequent pseudorotational analysis on ddI (1), ddA (2), ddG (3) and ddC (4) shows that the twist C2'exo-C3'-endo forms of sugar are overwhelmingly preferred (75-80%) over the C2'-endo envelope forms. The phase angles (P) for North and South conformers with the corresponding puckering amplitude (psi m) for ddI (1), ddA (2) and ddG (3) are as follows: PN = 0.1 degrees, PS = 161 degrees and psi m = 34.1 degrees for ddI (1); PN = 1.4 degrees, PS = 160 degrees and psi m = 34.2 degrees for ddA (2) and PN = 2.4 degrees, PS = 163 degrees and psi m = 33.6 degrees for ddG (3). The predominant North conformer of ddC (4) is intermediate between twist C2'-exo-C3'-endo and C3'-endo envelope (P = 10.9 degrees) with a psi m of 34.7 degrees. Note that these preponderant North-sugar structures (approx. 75-80%) found in the solution studies of ddI (1), ddA (2), dG (3) and ddC (4) are not reflected in the X-ray crystal structures of 2',3'-dideoxyadenosine and 2',3'-dideoxycytidine. The constituent sugar residues in both of these crystal structures denosine and 2',3'-dideoxycytidine. The constituent sugar residues in both of these crystal structures are found to be in the South-type geometry (ddA crystalizes in C3'-exo envelope form, while ddC adopts the form intermediate between the C3'-exo envelope and C3'-endo-C4'-exo twist form). This means that X-ray structures of ddA (2) and ddC (4) only represent the minor conformer of the overall pseudorotamer population in solution. An assumption that the structure of the pentofuranose sugar (i.e. P and psi m) participating in conformational equilibrium described by the two-state model remains unchanged at different temperatures has been experimentally validated by assessing five unknown pseudorotational parameters with eight unique observables (3J1'2', 3J1'2", 3J2'3', 3J2'3", 3J2"3', 3J2"3", 3J3'4' and 3J3"4') for 2',3'-dideoxynucleosides.(ABSTRACT TRUNCATED AT 400 WORDS)     

X-ray crystal structure of galabiose, O-alpha-D-galactopyranosyl-(1---4)-D-galactopyranose

O-alpha-D-Galactopyranosyl-(1---4)-D-galactopyranose, C12H22O11, Mr = 342.30, crystallises in the orthorhombic space group P2(1)2(1)2(1), and has alpha = 5.826(1), b = 13.904(3), c = 17.772(4) A, Z = 4, and Dx = 1.579 g.cm-3. Intensity data were collected with a CAD4 diffractometer. The structure was solved by direct methods and refined to R = 0.063 and Rw = 0.084 for 2758 independent reflections. The glycosidic linkage is of the type 1-axial-4-axial with torsion angles phi O-5' (O-5'-C-1'-O-1'-C-4) = 98.1(2) degrees, psi C-3 (C-3-C-4-O-1'-C-1') = -81.9(3) degrees, phi H (H-1'-C-1'-O-1'-C-4) = -18 degrees, and psi H (H-4-C-4-O-1'-C-1') = 35 degrees. The conformation is stabilised by an O-3 . . . O-5' intramolecular hydrogen-bond with length 2.787(3) A and O-3-H . . . O-5' = 162 degrees. The glycosidic linkage causes a folding of the molecule with an angle of 117 degrees between the least-square planes through the pyranosidic rings. The crystal investigated contained 56(1)% of alpha- and 44(1)% of beta-galabiose as well as approximately 70% of the gauche-trans and approximately 30% of the trans-gauche conformers about the exocyclic C-5'-C-6' and C-5-C-6 bonds. The crystal packing is governed by hydrogen bonding that engages all oxygen atoms except the intramolecular acceptor O-5' and the glycosidic O-1' oxygen atoms.     

Molecular and crystalline structure of 3'-methylamino-2',3'-dideoxyribosylthymine--a potential inhibitor of HIV]

The structure of 3'-methylamino-2',3'-dideoxyribosylthymine [ddT(3'NHMe)] was determined by X-ray analysis. The space group is P2(1)2(1)2(1). Cell dimensions are: a 5.132(1), b 13.718(1), c 16.947(2) A, V 1193.2 A3, Z 4. The structure was solved by directed methods and refined by the full-matrix least square method to R 4.8%. The molecule of ddT(3'NHMe) has anti-conformation with respect to the glycosidic bond (chi (O4'-C1'-N1-C2) = -106.7 degrees), C3'-endo-C4'-exo puckering of the sugar moiety (P -28.8 degrees, psi m -31.5 degrees) and gauche-gauche conformation about exocyclic C4'-C5' bond (psi(C3'-C4'-C5'-O5') 45.8 degrees). The structure of ddT(3'NHMe) was compared with those of 3'-amino-3'-deoxythymidine, 3'-azido-3'-deoxythymidine and natural thymidine.     

Photoinhibition, bleaching susceptibility and mortality in two scleractinian corals, Platygyra ryukyuensis and Stylophora pistillata, in response to thermal and light stresses

In the present study, we examined the effect of thermal stress on the photoinhibitory light threshold in a bleaching susceptible (Stylophora pistillata) and a bleaching resistant (Platygyra ryukyuensis) coral. Four light (0, 110, 520, 1015 micromol quantam(-2)s(-1)) and three temperature (26, 32 and 34 degrees C) conditions were used over a 3-h period, followed by 24- and 48-h recovery periods at approximately 21 degrees C under dim light. Dynamic photoinhibition could be detected in both P. ryukyuensis and S. pistillata under 520 and 1015 micromol quantam(-2)s(-1) at 26 degrees C and under 110 micromol quantam(-2)s(-1) at 32 degrees C only in S. pistillata. Chronic photoinhibition was recorded under 520 and 1015 micromol quantam(-2)s(-1) at 34 degrees C in P. ryukyuensis, and under 1015 micromol quantam(-2)s(-1) at 32 degrees C and under all light levels at 34 degrees C in S. pistillata. These results show that high temperature reduced the threshold light intensity for photoinhibition differently in two corals with different bleaching susceptibilities under thermal stress. No visual paling and mortality in P. ryukyuensis was observed at any treatment, even in chronically photoinhibited specimens, while paling and high mortality of S. pistillata was noted in all treatments, apart from samples at 26 degrees C. These observations suggest a potential role of the host in differential bleaching and mortality determination.     

Acyl migration on nucleic acid compounds related to adenosine

Treatment of N6,N6-di-p-toluyl-2',3'-O-isopropylideneadenosine (7) with ZnBr2 in 1,4-dioxane afforded a 8,5'-O-cycloadenosine derivative 8 exclusively. Reaction of 2',3'-O-isopropylideneadenosine (1) with p-cyanobenzoyl chloride in a CH2Cl2-Et3N mixture afforded a ring-cleaved compound 11 as the main product.     

葡萄糖氧化酶的有机相共价固定化

将葡萄糖氧化酶(GOD)在最适pH条件下冻干后,以戊二醛活化的壳聚糖为载体,分别在传统水相和1,4-二氧六环、乙醚、乙醇三种不同的有机相中进行共价固定化。通过比较水相固定化酶和有机相固定化酶的酶比活力、酶学性质及酶动力学参数,考察酶在有机相中的刚性特质对酶在共价固定化过程中保持酶活力的影响。结果表明,戊二醛浓度为0.1%、加酶量为80 mg/1 g载体、含水1.6%的1,4-二氧六环有机相固定化GOD与水相共价固定化GOD相比,酶比活力提高2.9倍,有效酶活回收率提高3倍;在连续使用7次后,1,4-二氧六环有机相固定化GOD的酶活力仍为相应水相固定化酶的3倍。在酶动力学参数方面,不论是表观米氏常数,最大反应速度还是转换数,1,4-二氧六环有机相固定化的GOD(Kmapp=5.63 mmol/L,Vmax=1.70μmol/(min.mgGOD),Kcat=0.304 s-1)都优于水相共价固定化GOD(Kmapp=7.33 mmol/L,Vmax=1.02μmol/(min.mg GOD),Kcat=0.221 s-1)。因此,相比于传统水相,GOD在合适的有机相中进行共价固定化可以获得具有更高酶活力和更优催化性质的固定化酶。该发现可能为酶蛋白在共价固定化时因构象改变而丢失生物活性的问题提供解决途径。     

DFT studies of the disaccharide, alpha-maltose: relaxed isopotential maps

The disaccharide, alpha-maltose, forms the molecular basis for the analysis of the structure of starch, and determining the conformational energy landscape as the molecule oscillates around the glycosidic bonds is of importance. Thus, it is of interest to determine, using density functionals and a medium size basis set, a relaxed isopotential contour map plotted as a function of the phi(H) and psi(H) dihedral angles. The technical aspects include the method of choosing the starting conformations, the choice of scanning step size, the method of constraining the specific dihedral angles, and the fitting of data to obtain well defined contour maps. Maps were calculated at the B3LYP/6-31+G( *) level of theory in 5 degrees intervals around the (phi(H),psi(H))=(0 degrees ,0 degrees ) position, out to approximately +/-30 degrees or greater, for gg-gg'-c, gg-gg'-r, gt-gt'-c, gt-gt'-r, tg-tg'-c, and tg-tg'-r conformers, as well as one-split gg(c)-gg'(r) conformer. The results show that the preferred conformation of alpha-maltose in vacuo depends strongly upon the hydroxyl group orientations ('c'/'r'), but the energy landscape moving away from the minimum-energy position is generally shallow and transitions between conformational positions can occur without the addition of significant energy. Mapped deviations of selected parameters such as the dipole moment; the C1-O1-C4', H1-C1-O1, and H4'-C4'-O1 bond angles; and deviations in hydroxymethyl rotamers, O5-C5-C6-O6, O5'-C5'-C6'-O6', C5-C6-O6-H, and C5'-C6'-O6'-H', are presented. These allow visualization of the structural and energetic changes that occur upon rotation about the glycosidic bonds. Interactions across the bridge are visualized by deviations in H(O2)...O3', H(O3')...O2, and H1...H4' distances and the H(O2)-O2-C2-C1 and H'(O3')-O3'-C3'-C4' hydroxyl dihedral angles.     

Future CO2 concentrations, though not warmer temperatures, enhance wheat photosynthesis temperature responses

The temperature dependence of C3 photosynthesis is known to vary according to the growth environment. Atmospheric CO2 concentration and temperature are predicted to increase with climate change. To test whether long-term growth in elevated CO2 and temperature modifies photosynthesis temperature response, wheat (Triticum aestivum L.) was grown in ambient CO2 (370 micromol mol(-1)) and elevated CO2 (700 micromol mol(-1)) combined with ambient temperatures and 4 degrees C warmer ones, using temperature gradient chambers in the field. Flag leaf photosynthesis was measured at temperatures ranging from 20 to 35 degrees C and varying CO2 concentrations between ear emergence and anthesis. The maximum rate of carboxylation was determined in vitro in the first year of the experiment and from the photosynthesis-intercellular CO2 response in the second year. With measurement CO2 concentrations of 330 micromol mol(-1) or lower, growth temperature had no effect on flag leaf photosynthesis in plants grown in ambient CO2, while it increased photosynthesis in elevated growth CO2. However, warmer growth temperatures did not modify the response of photosynthesis to measurement temperatures from 20 to 35 degrees C. A central finding of this study was that the increase with temperature in photosynthesis and the photosynthesis temperature optimum were significantly higher in plants grown in elevated rather than ambient CO2. In association with this, growth in elevated CO2 increased the temperature response (activation energy) of the maximum rate of carboxylation. The results provide field evidence that growth under CO2 enrichment enhances the response of Rubisco activity to temperature in wheat.     

Regioselective and stereoselective cleavage of cis-4-methyl-2-vinyl-l,3-dioxane by alkyllithiums

The reaction of alkyllithiums with cis-4-methyl-2-vinyl-1,3-dioxane proceeds in virtually quantitative yield via a formal S(N)2' ring opening that proceeds in a regioselective manner to give preferential cleavage of the C(2)-O(l) bond remote from the 4-methyl substituent, affording the E-enol ether as the major stereoisomer. A two-step mechanism, involving addition of the alkyllithium followed by syn-elimination of lithium alkoxide, is suggested to account for the stereochemical outcome.     

Synthesis and characterization of 6-O-beta-lactosyl-alpha,beta-lactoses, 1-O-(6-O-beta-lactosyl-beta-lactosyl)-(R,S)-glycerols, and 4,6-di-O-beta-D-galactopyranosyl-alpha,beta-D-glucoses.

1,2,3,2',3',4',6'-Hepta-O-acetyl-beta-lactose (4) was coupled with 2,3,6,2',3',4',6'-hepta-O-acetyl-alpha-lactosyl bromide (7) in the presence of Hg(CN)2 to afford 1,2,3,2',3',4',6'-hepta-O-acetyl-6-O-(2,3,6,2',3',4',6'-hepta-O-acetyl-b eta- lactosyl)-beta-lactose (11) which, upon O-deacetylation, gave 6-O-beta-lactosyl-alpha,beta-lactoses (64% from 4). In contrast, the reaction of 7 with benzyl 2,3,2',3',4',6'-hexa-O-acetyl-beta-lactoside in the presence of Hg(CN)2 produced 3,6,2',3',4',6'-hexa-O-acetyl-1,2-O- (2,3,2',3',4',6'-hexa-O-acetyl-1-O-benzyl-beta-lactos-6-yl orthoacetyl)-alpha-lactose (63%) and 3,6,2',3',4',6'-hexa-O-acetyl-1,2-O-(1- cyanoethylidene)-alpha-lactose (27%). The glycosidation of 4 using 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide in the presence of Hg(CN)2 afforded, after deprotection, 4,6-di-O-beta-D-galactopyranosyl-alpha,beta-D-glucoses (66%). The reaction of 11 with 1,2-di-O-benzyl-(R,S)-glycerols and trimethylsilyl trifluoromethanesulfonate yielded, after deprotection, 1-O-(6-O-beta-lactosyl-beta-lactosyl)-(R,S)-glycerols (18%). Under the same coupling conditions 11 reacted with 2-O-benzylglycerol to form 3-O-acetyl-2-O-benzyl-1-O-[2',3',4',6'-hexa-O-acetyl-6-O-(2,3,6,2',3',4' ,6'- hepta-O-acetyl-beta-lactosyl)-beta-lactosyl]-(R,S)-glycerols (16%).     

Synthesis and properties of 3'-amino-2',4'-BNA, a bridged nucleic acid with a N3'-->P5' phosphoramidate linkage

The synthesis and properties of a bridged nucleic acid analogue containing a N3'-->P5' phosphoramidate linkage, 3'-amino-2',4'-BNA, is described. A heterodimer containing a 3'-amino-2',4'-BNA thymine monomer, and thymine and methylcytosine monomers of 3'-amino-2',4'-BNA and their 5'-phosphoramidites, were synthesized efficiently. The dimer and monomers were incorporated into oligonucleotides by conventional 3'-->5' assembly, and 5'-->3' reverse assembly phosphoramidite protocols, respectively. Compared to a natural DNA oligonucleotide, modified oligonucleotides containing the 3'-amino-2',4'-BNA residue formed highly stable duplexes and triplexes with single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded DNA (dsDNA) targets, with the average increase in melting temperature (T(m)) against ssDNA, ssRNA and dsDNA being +2.7 to +4.0 degrees C, +5.0 to +7.0 degrees C, and +5.0 to +11.0 degrees C, respectively. These increases are comparable to those observed for 2',4'-BNA-modified oligonucleotides. In addition, an oligonucleotide modified with a single 3'-amino-2',4'-BNA thymine residue showed extraordinarily high resistance to nuclease degradation, much higher than that of 2',4'-BNA and substantially higher even than that of 3'-amino-DNA and phosphorothioate oligonucleotides. The above properties indicate that 3'-amino-2',4'-BNA has significant potential for antisense and antigene applications.     

The induction of hepatic microsomal UDP-glucuronosyltransferase by the methylsulfonyl metabolites of polychlorinated biphenyl congeners in rats

The effects of nine methylsulfonyl (MeSO(2)) metabolites of tetra-, penta- and hexachlorinated biphenyls (tetra-, penta- and hexaCBs; 20 micromol/kg once daily for 4 days) on the hepatic microsomal UDP-glucuronosyltransferase (UDP-GT) were investigated in male Sprague-Dawley rats. Each of the seven 3-MeSO(2)-PCBs, 3-MeSO(2)-2, 2',4',5-tetraCB (3-MeSO(2)-CB49), 3-MeSO(2)-2,3',4',5-tetraCB (3-MeSO(2)-CB70), 3-MeSO(2)-2,2',3',4',5-pentaCB (3-MeSO(2)-CB87), 3-MeSO(2)-2,2',4',5,5'-pentaCB (3-MeSO(2)-CB101), 3-MeSO(2)-2,2',3', 4',5,6-hexaCB (3-MeSO(2)-CB132), 3-MeSO(2)-2,2',3',4',5,5'-hexaCB (3-MeSO(2)-CB141), 3-MeSO(2)-2,2',4',5,5',6-hexaCB (3-MeSO(2)-CB149) and 4-MeSO(2)-2,2',4',5,5'-pentaCB (4-MeSO(2)-CB101) increased the activities of UDP-GT toward chloramphenicol, 4-nitrophenol and 4-methylumbelliferone. 4-MeSO(2)-2,2',4',5,5',6-hexaCB (4-MeSO(2)-CB149) increased the activity of UDP-GT toward chloramphenicol (UGT2B1) but not toward 4-nitrophenol (UGT1A6) and 4-methylumbelliferone (UGT1A6). The activity of UDP-GT toward thyroxine (T(4)) significantly increased after the administration of each of the seven 3-MeSO(2)-PCBs and 4-MeSO(2)-CB101. Significant correlation was found between the activity of UDP-GT toward T(4) and serum total T(4) concentration after the administration of each of the MeSO(2) derivatives except 4-MeSO(2)-CB149. In conclusion, seven 3-MeSO(2)-PCBs and 4-MeSO(2)-CB101 induce both UGT2B1 and UGT1A6, and 4-MeSO(2)-CB149 induces UGT 2B1. The results from the present study indicate that increase in the hepatic T(4) glucuronidation after the administration of the seven 3-MeSO(2)-PCBs and 4-MeSO(2)-CB101 possibly because of the induction of both UGT1A1 and UGT1A6 caused the reduction of serum T(4) levels.     

Molecular structure of deoxyadenylyl-3'-methylphosphonate-5'-thymidine dihydrate, (d-ApT x 2H2O), a dinucleoside monophosphate with neutral phosphodiester backbone. An X-ray crystal study.

dApT, a modified deoxyribose dinucleoside phosphate with an uncharged methylphosphonate group, crystallizes as dihydrate in space group P2(1)2(1)2, a = 9.629(3), b = 20.884(6) and c = 14.173(4)A, Z = 4. The structure has been determined using 2176 X-ray diffractometer reflections and refined to a final R of 0.105. Torsion angles about P-O(5') and P-O(3') bonds are -91.8 degrees and 117.8 degrees. The former is in the normal (-)gauche range while the latter is eclipsed. Bases are oriented anti, the sugar of adenosine is puckered 2T3 (C(2')endo) whereas that of thymidine displays puckering disorder with major and minor occupancy sites. Major site is a half-chair 2T (C(2')endo-C(1')exo) and minor site an envelope 3T2 (C(3(1)endo). Adenine and thymine bases of symmetry related molecules form reversed Hoogsteen type base pairs, water molecules are disordered in the crystal lattice.     

Investigations on the dynamic structures of adenine- and thymine-containing DNA.

The structures of poly(dA-dT), poly(dA-dBr5U) and of poly(dA).poly(dT) have been investigated in solution and in fibers, by Raman spectroscopy. Both the alternating poly(dA-dT), poly(dA-dBr5U) and non-alternating poly(dA).poly(dT) exhibit, in the region of sugar phosphate backbone vibrations, two bands of almost equal intensity at about 841 cm-1 and 817 cm-1. The analysis of the characteristic bands of thymine residues that are sensitive to sugar puckers gives indication of a significant displacement from the C(2')-endo conformer suggesting the adoption of alternative conformers such as O(4')-endo. In contrast, the diagnostic Raman bands for the sugar pucker of adenine residues suggest, instead, predominant adoption of C(2')-endo conformations. These Raman results are compatible with rapid dynamic changes of sugar puckers between C(2')-endo and O(4')-endo for the thymidine (and uridine) residues, whereas in adenine residues the sugar puckers fluctuate around the C(2')-endo pucker in all synthetic DNA molecules studied. Molecular dynamics simulations, performed on six different starting models using two distance-dependent dielectric functions epsilon(r) = 4 r and a sigmoidal dependence), all gave similar dynamic behavior in agreement with these Raman data and their interpretation. The mean calculated pseudorotation phases of the adenine residues are systematically higher (around C(2')-endo) than those of the thymine residues (close to O(4')-endo-C(1')-exo). Besides, the mean lifetimes of the thymine residues are 1.5 to 2.0-fold higher in the O(4')-endo than in the C(2')-endo domain, while those of the adenine residues are two to threefold higher in the C(2')-endo than in the O(4')-endo domain. In the Raman spectra of the alternating poly(dA-dBr5U), the splitting of a band into two components arising from the two contributions of ApBr5U and Br5UpA provides strong evidence for a repeating dinucleotide structure in solution. The calculated twist values averaged over the simulation runs are also systematically higher in the 5'T-A3' step (39 degrees) than in the 5'A-T3' step (33 degrees). Simultaneously, the calculated roll values are positive in the 5'T-A3' step (6 degrees) and negative in the 5'A-T3' step (-9 degrees), while the propeller twist values are about the same (-11 degrees to -16 degrees). On the other hand, in the homopolymer, the average twist value is close to 36 degrees with the roll angle close to 0 degrees and large propeller twist values (-20 degrees).     

Design of peptides with branched beta-carbon dehydro-residues: syntheses, crystal structures and molecular conformations of two peptides, (I) N-Carbobenzoxy-DeltaVal-Ala-Leu-OCH3 and (II) N-Carbobenzoxy-DeltaIle-Ala-Leu-OCH3.

Highly specific structures can be designed by inserting dehydro-residues into peptide sequences. The conformational preferences of branched beta-carbon residues are known to be different from other residues. As an implication it was expected that the branched beta-carbon dehydro-residues would also induce different conformations when substituted in peptides. So far, the design of peptides with branched beta-carbon dehydro-residues at (i + 1) position has not been reported. It may be recalled that the nonbranched beta-carbon residues induced beta-turn II conformation when placed at (i + 2) position while branched beta-carbon residues induced beta-turn III conformation. However, the conformation of a peptide with a nonbranched beta-carbon residue when placed at (i + 1) position was not found to be unique as it depended on the stereochemical nature of its neighbouring residues. Therefore, in order to induce predictably unique structures with dehydro-residues at (i + 1) position, we have introduced branched beta-carbon dehydro-residues instead of nonbranched beta-carbon residues and synthesized two peptides: (I) N-Carbobenzoxy-DeltaVal-Ala-Leu-OCH3 and (II) N-Carbobenzoxy-DeltaIle-Ala-Leu-OCH3 with DeltaVal and DeltaIle, respectively. The crystal structures of peptides (I) and (II) have been determined and refined to R-factors of 0.065 and 0.063, respectively. The structures of both peptides were essentially similar. Both peptides adopted type II beta-turn conformations with torsion angles; (I): phi1 = -38.7 (4) degrees, psi1 = 126.0 (3) degrees; phi2 = 91.6 (3) degrees, psi2 = -9.5 (4) degrees and (II): phi1 = -37.0 (6) degrees, psi1 = 123.6 (4) degrees, phi2 = 93.4 (4), psi2 = -11.0(4) degrees respectively. Both peptide structures were stabilized by intramolecular 4-->1 hydrogen bonds. The molecular packing in both crystal structures were stabilized in each by two identical hydrogen bonds N1...O1' (-x, y + 1/2, -z) and N2...O2' (-x + 1, y + 1/2, -z) and van der Waals interactions.     

Effect of pH on the ternary solution behavior of beta-lactoglobulin

Ternary phase diagrams (TPDs) were constructed for aqueous beta-lactoglobulin solutions containing ethanol and (NH4)2SO4 at pHs of 7, 5, and 3 for temperatures between 20 and 70 degrees C. The addition of (NH4)2SO4 generally led to the production of a reversible precipitate, a transformation that was not strongly influenced by temperature or pH. In contrast, at pH 7 and 20 degrees C, ethanol concentrations >12% led to the formation of a molten-globule structure, which gelled at protein concentrations >10%. Destabilization of beta-lactoglobulin structure occurred at lower ethanol concentrations as temperature was increased, until at 70 degrees C, all solutions that were previously liquid at room temperature had transformed into a gel. At pH 5.0, near beta-lactoglobulin's isoelectric point, demixing dominated, leading to the creation of either irreversible precipitates or a paste-like microgel. Elevated temperatures caused the previously liquid morphology to transform into either a reversible aggregate or microgel. Solution behavior at pH 3 had characteristics of what was observed at pHs 7 and 5. At moderated protein and ethanol concentrations, a paste-like microgel was observed, whereas at higher ethanol concentrations, beta-lactoglobulin formed a gel. This work demonstrates how small changes in protein structure at the molecular level can have a dramatic effect on macroscopic morphology.     

Purification and properties of Bacillus subtilis SA-22 endo-1, 4-beta-D-mannanase.

beta-mannanase (EC 3.2.1.78) from Bacillus subtilis SA-22 was purified successively by ammonium sulfate precipitation, hydroxyapatite chromatography, Sephadex G-75 gel filtration and DEAE-52 anion-exchange chromatography. Through these steps, the enzyme was concentrated 30.75-fold with a recovery rate of 23.43%, with a specific activity of 34780.56 u/mg. Molecular weight of the enzyme was determined to be 38 kD by SDS-PAGE and 34 kD by gel filtration. The results revealed that the optimal pH value for the enzyme was 6.5 and the optimal temperature was 70 degrees C. The enzyme is stable between pH 5 to 10. The enzyme remained most of its activity after a treatment of 4 h at 50 degrees C, but lost 25% of activity at 60 degrees C for 4 h, lost 50% of activity at 70 degrees C for 3 h. The enzyme activity was strongly inhibited by Hg2+. The Michaelis constants (Km) were measured as 11.30 mg/mL for locust bean gum and 4.76 mg/mL for konjac powder, while Vmax for these two polysaccharides were 188.68 (micromol x mL(-1) x min(-1)) and 114.94 (micromol x mL(-1) x min(-1)), respectively.