Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea.

Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.     

Salt induced B----A transition of poly(dG).poly(dC) and the stabilization of A form by its methylation.

Raman spectra of poly(dG).poly(dC) have been observed in aqueous solutions at various ionic strengths, [NaCl] = 0.03 to 4 M, and at different temperatures, 10 to 60 degrees C. At 30 degrees C, and at [NaCl] = 0.03 M, it was found to have a B-form (with O4'endo-anti guanosine and C2'endo-anti cytidine), whereas, at [NaCl] = 4 M, an A form (with C3'endo-anti guanosine and C3'endo-anti cytidine). At 30 degrees C and [NaCl] = 1 M, namely at an intermediate state, a fraction of this molecules was considered to have a "heteronomous A" form (with O4'endo-anti guanosine and C3' endo-anti cytidine). At 60 degrees C and [NaCl] = 1 M, it assumes the B form, and at 10 degrees C and [NaCl] = 1 M, the A form. Cytosine-5-methylation was found to cause a marked stabilization of the A form. Even at [NaCl] = 0.1 M (at 30 degrees C), a substantial portion of poly(dG).poly(dm5C) was found to have a heteronomous form, in which the dG atrand is in the B form and the dC an A form; it never assumes a complete B form.     

Field and in vitro assay of three methods for freezing ram semen

Glycerol has been the most widely used cryopreservation agent for spermatozoa and a wide range of factors affect its action on sperm viability and fertilizing capacity. We tested three methods for freezing ram semen packed in 0.25 ml straws (final cellular concentration: 100 x 10(6) spz/ml). Method M1: Two-thirds of the final volume of diluent was added as solution A (without glycerol) to the pure semen at 35 degrees C. The sample was cooled to 5 degrees C (-0.30 degrees C/min), one-third of final diluent volume was added as solution B (final concentration of glycerol 4%) and the sample was maintained at 5 degrees C for 2h. It was then frozen in a programmable biofreezer (-20 degrees C/min down to -100 degrees C). Method M2: The sample was diluted with a specific solution at 35 degrees C (final concentration of glycerol 3%), cooled to 5 degrees C (-0.20 degrees C/min) and left for 2h. After that, it was frozen in nitrogen vapours. Method M3: Semen was diluted 1:1 in a specific solution (concentration of glycerol 2%) and cooled to 5 degrees C (-0.25 degrees C/min). The sample was then diluted again in the same solution to the final cellular concentration (final concentration of glycerol 4%). It was left for 1h at 5 degrees C and then frozen in a programmable biofreezer (-20 degrees C/min down to -100 degrees C). Best total motility (TM) and progressive motility (PM) (75.8 and 55.18%) were obtained using Method M3. Methods M1 and M3 gave significantly higher values (P<0.05) for kinetic parameters: average path velocity (VAP) (81.3 and 85.2 microm/s), straight-line velocity (VSL) (72.8 and 77.3 microm/s) and linearity (LIN) (66.6 and 68.8%). Method M2 showed the lowest kinetic parameters of motility (VAP 74.4, VSL 67.3 and LIN 62.5) and the highest percentage of cells with damaged plasma membrane (53.8%). Method M1 gave the worst results in viability and acrosome status assessed using fluorescence probes (31.3%-dead cells with damaged acrosomes-versus 25.4% in M2 and 23.3% in M3). A field trial carried out on fertility showed a significantly higher percentage of pregnant or lambing ewes (P<0.05) with Method M3 (67.3% versus 51.1% for M1 and 58.8% for M2). We concluded that the use of a simple dilution medium (test-fructose-glycerol-egg yolk) with the addition of glycerol (to 2% at 35 degrees C and to 4% at 5 degrees C) in two steps together with a programmable biofreezer was a productive method for freezing ram semen.     

Na2CO3 influence on DNA double helix stability: strong anion destabilizing effect

Addition of Na(2)CO(3) to almost salt-free DNA solution (5.10(-5)M EDTA, pH=5.7, T(m)=26.5 degrees C) elevates both pH and the DNA melting temperature (T(m)) if Na(2)CO(3) concentration is less than 0.004 M. For 0.004 M Na(2)CO(3), T(m)=58 degrees C is maximal and pH=10.56. Further increase in concentration gives rise to a monotonous decrease in T(m) to 37 degrees C for 1M Na(2)CO(3) (pH=10.57). Increase in pH is also not monotonous. The highest pH=10.87 is reached at 0.04 M Na(2)CO(3) (T(m)=48.3 degrees C). To reveal the cause of this DNA destabilization, which happens in a narrow pH interval (10.56/10.87) and a wide Na(2)CO(3) concentration interval (0.004/1M), a procedure has been developed for determining the separate influences on T(m) of Na(+), pH, and anions formed by Na(2)CO(3) (HCO(3)(-) and CO(3)(2-)). Comparison of influence of anions formed by Na(2)CO(3) on DNA stability with Cl(-) (anion inert to DNA stability), ClO(4)(-) (strong DNA destabilizing "chaotropic" anion) and OH(-) has been carried out. It has been shown that only Na(+) and pH influence T(m) in Na(2)CO(3) solution at concentrations lower than 0.001 M. However, the T(m) decrease with concentration for [Na(2)CO(3)]>/=0.004 M is only partly caused by high pH=10.7. Na(2)CO(3) anions also exert a strong destabilizing influence at these concentrations. For 0.1M Na(2)CO(3) (pH=10.84, [Na(+)]=0.2M, T(m)=42.7 degrees C), the anion destabilizing effect is higher 20 degrees C. For NaClO(4) (ClO(4)(-) is a strong "chaotropic" anion), an equal anion effect occurs at much higher concentrations approximately 3M. This means that Na(2)CO(3) gives rise to a much stronger anion effect than other salts. The effect is pH dependent. It decreases fivefold at neutral pH after addition of HCl to 0.1M Na(2)CO(3) as well as after addition of NaOH for pH greater than 11.2.     

Interactions between neutrophil gelatinase-associated lipocalin and natural lipophilic ligands

Neutrophils are activated by both paracrine molecules, e.g. platelet activating factor (PAF) and leukotriene B4 (LTB4), and the bacterial hydrophobic peptide N-formyl-Met-Leu-Phe (fMLP). Several mechanisms are involved in regulation of the activation, including receptor endocytosis and ligand breakdown. The interactions between the specific granule protein neutrophil gelatinase-associated lipocalin (NGAL), expressed in human neutrophils, and fMLP, PAF and LTB4, were investigated by weak affinity chromatography. NGAL was immobilised to a silica matrix and packed in a micro-column and the retention times of retarded ligands were measured and used to calculate the strength of the interactions. The association constants for fMLP were K(ass) = 0.85 x 10(3) M(-1) at 20 degrees C and 0.77 x 10(3) M(-1) at 37 degrees C, for LTB4 were K(ass) = 4.37 x 10(3) M(-1) at 20 degrees C and 3.27 x 10(3) M(-1) at 37 degrees C and for PAF were K(ass) = 25.4 x 10(3) M(-1) at 20 degrees C and 10.5 x 10(3) M(-1) at 37 degrees C. Other methods of detecting the interactions such as gel filtration, immunoprecipitation, photoactivated ligands and fluorescence quenching proved to be insufficient. The results demonstrate the superiority of weak affinity chromatography as a method of studying the interactions of the specific granule protein NGAL.     

Water temperature and intensity of exercise in maintenance of thermal equilibrium

This study examined the thermal and metabolic responses of six men during exercise in water at critical temperature (Tcw, 31.2 +/- 0.5 degrees C), below Tcw (BTcw, 28.8 +/- 0.6 degrees C), at thermoneutrality (Ttn, 34 degrees C), and above Ttn (ATtn, 36 degrees C). At each water temperature (Tw) male volunteers wearing only swimming trunks completed four 1-h experiments while immersed up to the neck. During one experiment, subjects remained at rest (R), and the other three performed leg exercise (LE) at three different intensities (LE-1, 2 MET; LE-2, 3 MET; LE-3, 4 MET). In water warmer than Tcw, there was no difference in metabolic rate (M) during R. The M for each work load was independent of Tw. Esophageal temperature (Tes) remained unchanged during R in water of ATtn (36 degrees C). However, Tes significantly (P less than 0.05) declined over 1 h during R at Ttn (delta Tes = -0.39 degrees C), Tcw (delta Tes = -0.54 degrees C), and BTcw (delta Tes = -0.61 degrees C). All levels of underwater exercise elevated Tes and M compared with R at all Tw. In water colder than Tcw, the ratio of heat loss from limbs compared with the trunk became greater as LE intensity increased, indicating a preferential increase in heat loss from the limbs in cool water. Tissue insulation (Itissue) was lower during LE than at R and was inversely proportional to the increase in LE intensity. A linearly inverse relationship was established between Tw and M in maintaining thermal equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affinity (Kd = 10(-9) M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [3H]tamoxifen binding capacity is thermolabile, being most stable at 4 degrees C and rapidly lost at 37 degrees C. More [3H]tamoxifen, however, is specifically bound at incubation temperatures of 25 degrees C and 37 degrees C than at 4 degrees C although prewarming nuclei has no effect, suggesting exchange of [3H]tamoxifen for an unidentified endogeneous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (Kd = 10(-9) M) [3H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78 +/- 0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000 X g, 30 min) contains high-capacity (955 +/- 405 (S.D.) fmol/mg protein), low-affinity (Kd = 10.9 +/- 4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000 X g, 60 min) contains low-capacity (46 +/- 15 (S.D.) fmol/mg protein), high-affinity (Kd = 0.61 +/- 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%-3.2%, and nuclear sites less than 0.5% of total sites.     

The synthesis and properties of oligodeoxyribonucleotides containing N6-methoxyadenine.

Oligodeoxyribonucleotides containing N6-methoxyadenine (M) have been synthesized. The order of stability of duplexes consisting of synthesized oligodeoxyribonucleotides, 5'd(CCTGGTAXCAGGTCC)3'-5'd(GGACCTGNTACCAGG)3' (X = M, A, G. N = A, G, T, C), was M: A (Tm = 52 degrees C) greater than M: T (50 degrees C) greater than M: G (48 degrees C) greater than M: C (46 degrees C) observed by thermal denaturation in a buffer of 0.01 M Na cacodylate, and 0.1 M NaCl at pH 7.0. The Tms are within a range of 6 degrees of difference, which is smaller than those of Tms of the duplexes containing A:N pairs (11 degrees) and G:N pairs (11 degrees). DNA replication study on a template-primer system, 5'd(32p-CAGCTTTCGC)3' 3'd(GTCGAAAGCGMAGTCG)5', showed that TTP and dCTP were incorporated into DNA strands at a site opposite to M by Klenow DNA polymerase, but dATP and dGTP were not.     

Structure and metastability of N-lignocerylgalactosylsphingosine (cerebroside) bilayers

Differential scanning calorimetry (DSC) and X-ray diffraction have been used to study hydrated N-lignocerylgalactosylsphingosine (NLGS) bilayers. DSC of fully hydrated NLGS shows an endothermic transition at 69-70 degrees C, immediately followed by an exothermic transition at 72-73 degrees C; further heating shows a high-temperature (Tc = 82 degrees C), high-enthalpy (delta H = 15.3 kcal/mol NLGS) transition. Heating to 75 degrees C, cooling to 20 degrees C and subsequent reheating shows no transitions at 69-73 degrees C; only the high-temperature (82 degrees C), high-enthalpy (15.3 kcal/mol) transition. Two exothermic transitions are observed on cooling; for the upper transition its temperature (about 65 degrees C) and enthalpy (about 6 kcal/mol NLGS) are essentially independent of cooling rate, whereas the lower transition exhibits marked changes in both temperature (30----60 degrees C) and enthalpy (2.2----9.5 kcal/mol NLGS) as the cooling rate decreases from 40 to 0.625 Cdeg/min. On reheating, the enthalpy of the 69-70 degrees C transition is dependent on the previous cooling rate. The DSC data provide clear evidence of conversions between metastable and stable forms. X-ray diffraction data recorded at 26, 75 and 93 degrees C show clearly that NLGS bilayer phases are present at all temperatures. The X-ray diffraction pattern at 75 degrees C shows a bilayer periodicity d = 65.4 A, and a number of sharp reflections in the wide-angle region indicative of a crystalline chain packing mode. This stable bilayer form converts to a liquid-crystal bilayer phase; at 93 degrees C, the bilayer periodicity d = 59.1 A, and a diffuse reflection at 1/4.6 A-1 is observed. The diffraction pattern at 22 degrees C represents a combination of the stable and metastable low-temperature bilayer forms. NLGS exhibits a complex pattern of thermotropic changes related to conversions between metastable (gel), stable (crystalline) and liquid-crystalline bilayer phases. The structure and thermotropic properties of NLGS are compared with those of hydrated N-palmitoylgalactosylsphingosine reported previously (Ruocco, M.J., Atkinson, D., Small, D.M., Skarjune, R.P., Oldfield, E. and Shipley, G.G. (1981) Biochemistry 20, 5957-5966).     

Equilibrium analysis of binding of Candida albicans to human buccal epithelial cells

The effect of growth temperature on the binding of Candida albicans to human buccal epithelial cells (BECs) was examined using an equilibrium of binding analysis. Candida albicans was cultured in M9 medium either for 12 h at 25 degrees C or for 9 h at 25 degrees C and then shifted to 37 degrees C for 3 h. The temperature shift did not result in germ tube formation; however, the adherence of C. albicans to BECs was altered. Shifting temperature increased the yeast's ability to bind to BECs. A Langmuir adsorption isotherm was used to calculate the maximum number of available binding sites (N) and the apparent association constants of binding (Ka) for all resolvable adhesin-receptor interactions. Three classes of adhesin-receptor interactions were resolved when the yeast was cultured at 25 degrees C and included a low copy number site (N = 3.0 cfu/BEC; Ka = 2.11 X 10(-6) mL/cfu), a medium copy number site (N = 23.6 cfu/BEC, Ka = 8.21 X 10(-7) mL/cfu), and a high copy number site (N = 91.7 cfu/BEC, Ka = 3.35 X 10(-8) mL/cfu). Two classes of adhesin-receptor interactions were resolved when the incubation temperature was shifted to 37 degrees C: a low copy number site (N = 4.5 cfu/BEC, Ka = 3.98 X 10(-6) mL/cfu) and a high copy number site (N = 150.5 cfu/BEC, Ka = 8.47 X 10(-8) mL/cfu). Augmented C. albicans adherence to BECs due to the elevated growth temperatures appears to result from a temperature-regulated alteration in the C. albicans adhesin that recognizes a high copy number receptor site with relatively low affinity.     

Changes in thermal insulation during underwater exercise in Korean female wet-suit divers

The present work was undertaken to examine the effect of wet suits on the pattern of heat exchange during immersion in cold water. Four Korean women divers wearing wet suits were immersed to the neck in water of critical temperature (Tcw) while resting for 3 h or exercising (2-3 met on a bicycle ergometer) for 2 h. During immersion both rectal (Tre) and skin temperatures and O2 consumption (VO2) were measured, from which heat production (M = 4.83 VO2), skin heat loss (Hsk = 0.92 M +/- heat store change based on delta Tre), and thermal insulation were calculated. The average Tcw of the subjects with wet suits was 16.5 +/- 1.2 degrees C (SE), which was 12.3 degrees C lower than that of the same subjects with swim suits (28.8 +/- 0.4 degrees C). During the 3rd h of immersion, Tre and mean skin temperatures (Tsk) averaged 37.3 +/- 0.1 and 28.0 +/- 0.5 degrees C, and skin heat loss per unit surface area 42.3 +/- 2.66 kcal X m-2 X h. The calculated body insulation [Ibody = Tre - Tsk/Hsk] and the total shell insulation [Itotal = (Tre - TW)/Hsk] were 0.23 +/- 0.02 and 0.5 +/- 0.04 degrees C X kcal-1 X m2 X h, respectively. During immersion exercise, both Itotal and Ibody declined exponentially as the exercise intensity increased. Surprisingly, the insulation due to wet suit (Isuit = Itotal - Ibody) also decreased with exercise intensity, from 0.28 degrees C X kcal-1 X m2 X h at rest to 0.12 degrees C X kcal-1 X m2 X h at exercise levels of 2-3 met.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of in vitro matured,in vitro fertilized domestic cat embryos following cryopreservation,culture and transfer

The ability of embryos to successfully survive cryopreservation is dependent on both morphological and developmental characteristics. Domestic cat oocytes matured in vitro exhibit alterations in nuclear and cytoplasmic maturation that may affect developmental competence, particularly after cryopreservation. In Experiment 1, we evaluated the developmental competence of in vitro produced (IVM/IVF) cat embryos after cryopreservation on Days 2, 4 or 5 of IVC. In Experiment 2, in vivo viability was examined by transfer of cryopreserved embryos into recipient queens. Oocytes recovered from minced ovaries were cultured in TCM-199 with hCG/eCG and EGF at 38 degrees C in 5% O(2), 5% CO(2), 90% N(2) for 24h. In Experiment 1, after IVM/IVF, on Day 2 (n=56), Day 4 (n=48) and Day 5 (n=42) of IVC, embryos were equilibrated for 10 min at 22 degrees C in HEPES (15m M) Tyrode's (HeTy) with 1.4M propylene glycol (PG), 0.125 M sucrose (S), 10% dextran and 10% FBS, loaded into 0.25 ml straws, cooled at 2.0 degrees C/min to -6.0 degrees C and held for 10 min. After seeding, cooling resumed at 0.3 degrees C/min to -30 degrees C and after a 10 min hold, straws were plunged into liquid nitrogen (LN(2)). Straws were thawed in air for 2 min and cryoprotectant was removed by a five-step rinse consisting of 3 min each in HeTY with 0.95 M PG/0.25 M S; 0.95 M PG/0.125 M S; 0.45 M PG/0.125 M S; 0 PG/0.125 M S; 0 PG/0.0625 M S. Contemporary IVM/IVF embryos were used as nonfrozen controls (Day 2, n=14; Day 4, n=26; Day 5, n=35). After 8 days of IVC, the number of embryos developing to blastocysts was recorded and blastocyst cell numbers were counted after staining with Hoechst 33342. In Experiment 1, developmental stage did not affect the survival rate after thawing (Day 2=79%, Day 4=90%, Day 5=98%) and was not different from that of controls (Day 2=89%, Day 4=88%, Day 5=96%). Blastocyst development was similar among days both after cryopreservation (Day 2=59%, Day 4=54%, Day 5=63%) and in controls (Day 2=55%, Day 4=54%, Day 5=58%). Mean (+/-S.D.) cell number of blastocysts was slightly lower (NS) in cryopreserved embryos (Day 2=152+/-19, Day 4=124+/-20, Day 5=121+/-24) than in controls (Day 2=141+/-25, Day 4=169+/-21, Day 5=172+/-19). In Experiment 2, embryos frozen on Day 2 (n=68), Day 4 (n=49) or Day 5 (n=73) were thawed and cultured for 3, 1, or 0 days before transfer by laparotomy to 5 (mean=12.6+/-2.4), 4 (mean=12.2+/-3.7) and 6 (mean=12.0+/-1.6) recipients, respectively. Four recipients were pregnant on Day 21; two from embryos frozen on Day 4 and two from Day 5. Two live kittens were born at 66 days, a third kitten died during parturition at 64 days and a fourth pregnancy aborted by Day 45. In summary, we have shown that a controlled rate cryopreservation technique can be successfully applied to cat embryos produced by IVM/IVF. In vitro development to the blastocyst stage was not affected by the age of embryos at cryopreservation. The births of live kittens after ET of cryopreserved embryos is additional validation of progress toward applying assisted reproductive technology to preservation of endangered felids.     

X-ray diffraction and calorimetric study of N-lignoceryl sphingomyelin membranes.

Differential scanning calorimetry and x-ray diffraction have been used to investigate hydrated multibilayers of N-lignoceryl sphingomyelin (C24:0-SM) in the hydration range 0-75 wt % H2O. Anhydrous C24:0-SM exhibits a single endothermic transition at 81.3 degrees C (delta H = 3.6 kcal/mol). At low hydration (12.1 wt % H2O), three different endothermic transitions are observed: low-temperature transition (T1) at 39.4 degrees C (transition enthalpy (delta H1) = 2.8 kcal/mol), intermediate-temperature transition (T2) at 45.5 degrees C, and high-temperature transition (T3) at 51.3 degrees C (combined transition enthalpy (delta H2 + 3) = 5.03 kcal/mol). On increasing hydration, all three transition temperatures of C24:0-SM decrease slightly to reach limiting values of 36.7 degrees C (T1), 44.4 degrees C (T2), and 48.4 degrees C (T3) at approximately 20 wt % H2O. At 22 degrees C (below T1), x-ray diffraction of C24:0-SM at different hydration levels shows two wide-angle reflections, a sharp one at 1/4.2 A-1 and a more diffuse one at 1/4.0 A-1 together with lamellar reflections corresponding to bilayer periodicities increasing from d = 65.4 A to a limiting value of 71.1 A. Electron density profiles show a constant bilayer thickness dp-p approximately 50 A. In contrast, at 40 degrees C (between T1 and T2) a single sharp wide-angle reflection at approximately 1/4.2 A-1 is observed. The lamellar reflections correspond to a larger bilayer periodicity (increasing from d = 69.3-80.2 A) and there is some increase in dp-p (52-56 A) with hydration.(ABSTRACT TRUNCATED AT 250 WORDS)     

The two modes of binding of Ru(phen)(2)dppz(2+) to DNA: thermodynamic evidence and kinetic studies

The binding of Ru(phen)(2)dppz(2+) (dppz=dipyrido[3,2-a:2',3'-c]phenazine) to DNA was investigated at pH 7.0 and 25 degrees C using stopped-flow and spectrophotometric methods. Equilibrium measurements show that two modes of binding, whose characteristics depend on the polymer to dye ratio (C(P)/C(D)), are operative. The binding mode occurring for values of C(P)/C(D) higher than 3 exhibits positive cooperativity, which is confirmed by kinetic experiments. The reaction parameters are K=2 x 10(3)M(-1), omega=550, n=1, k(r)=(1.9+/-0.5) x 10(7)M(-1)s(-1) and k(d)=(9.5+/-2.5)x10(3)s(-1) at I=0.012 M. The results are discussed in terms of prevailing surface interaction with DNA grooves accompanied by partial intercalation of the dppz residue. The other binding mode becomes operative for C(P)/C(D)<3 and the equilibria analysis shows this is an ordinary intercalation mode (K=1.3 x 10(6) M(-1), n=1.5 at I=0.012 M and K=2 x 10(5) M(-1), n=1.2 at I=0.21 M). Similar behaviour is displayed by double-stranded poly(A).     

Asymmetric epoxidation of alpha-olefins having neighboring sugar chiral templates and alternating copolymerization with dicarboxylic anhydrides

Sugar-substituted epoxides 5-8 were synthesized by asymmetric epoxidation (in CH(2)Cl(2)/water) of alpha-olefins having neighboring sugars (1-4) by use of an achiral oxidant (MCPBA), in which the sugar moiety acted as a chiral template. The diastereoselectivities depend on the methylene spacer between vinyl group and carbohydrate derivatives. The methylene spacer between sugar and vinyl groups influenced the diastereoselectvity. In the case of epoxidation of 4 at 27 degrees C for 24 h, the diastereoselectivity was the highest (99/1). Copolymerizations of 5-8 with succinic anhydride were attained at 100 degrees C for 72 h to give poly(ethylene succinate) having pendant carbohydrate [poly(SAn-alt-5), M(n) = 1.4 x 10(3); poly(SAn-alt-6), M(n) = 2.2 x10(3); poly(SAn-alt-7), M(n) = 2.9 x 10(3); poly(SAn-alt-8), M(n) = 1.8 x 10(3)]. The methylene spacer between sugar and epoxide has an effect on the reactivity of epoxide in copolymerization as well as the diastereoselectivity. Alternating copolymerization of 7 and glutaric anhydride gave a polyester of M(n) 4.2 x10(3).     

DNA helicase RepA: cooperative ATPase activity and binding of nucleotides

The steady-state kinetic parameters of the ATPase activity of the homohexameric DNA helicase RepA and the binding of the fluorescent analogue epsilonADP to RepA have been studied. ssDNA stimulates RepA ATPase activity optimally at acidic pH 5.3-6.0. The sigmoidal kinetic curves in both the absence and presence of ssDNA show strong positive cooperativity for ATP hydrolysis, with oligonucleotides longer than 10mer optimal for ssDNA-stimulated ATPase activity. Fluorescence titrations show that, at 25 degrees C and in the absence of DNA, the binding of epsilonADP to RepA is biphasic with three high (K(1) = 1.54 x 10(6) M(-1)) and three low (K(2) = 4.71 x 10(4) M(-)(1)) affinity binding sites differing by 30-40-fold in binding constants. In the absence of cofactors, RepA melts cooperatively at T(m) = 65.8 +/- 0.1 degrees C and is more stable in the presence of ATPgammaS, T(m) = 68.1 +/- 0.2 degrees C (DeltaDeltaG 0.95 kcal/mol), than in the presence of ADP, T(m) = 66. 5 +/- 0.1 degrees C (DeltaDeltaG 0.29 kcal/mol), indicating that the additional phosphate group in ATPgammaS has a significant influence on RepA structure. A model is proposed in which individual subunits of RepA sequentially and cooperatively perform a multistep ATP hydrolytic cycle.     

Chemical structure and chain conformation of the water-insoluble glucan isolated from Pleurotus tuber-regium

A water-insoluble polysaccharide (TM8) was isolated from sclerotium of Pleurotus tuber-regium by extraction with 0.5M NaOH aqueous solutions at 120 degrees C. Its chemical structure was confirmed by infrared, high performance liquid chromatography, gas chromatography, and (13)C NMR in dimethylsulfoxide (DMSO) to be composed of beta-(1 --> 3)-D-glucan backbone chain linked with a branched glucose, one out of every three glycosyl units being substituted at C6 position. The glucan TM8 in DMSO was fractionated by nonsolvent addition method into ten fractions, and the solution properties were studied by size exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS) and viscometry in DMSO at 30 degrees C. The dependencies of intrinsic viscosity [eta] and radius of gyration [(s(2)(1/2)(z-2)] on weight-average molecular mass M(w) for this glucan were found to be [eta] = (9.24 +/- 0.2) x 10(-2)M(w)(0.51 +/- 0.02) (cm(3)g(-1)) and [(s(2)(1/2)(z-2)] = (3.67 +/- 0.3) x 10(-2)M(w)(0.56 +/- 0.02) (nm) in the range of M(w) from 1.07 x 10(4) to 77.4 x 10(4). Based on current theories for a wormlike chain, the conformational parameters of the glucan TM8 were found to be 408 (nm(-1)) for M(L), 3.1 (nm) for q, and 16.8 for C(infinity), suggesting that the polysaccharide exists as a dense random-coil chain in DMSO, due to branched structure.     

Role of galactomannan composition on the binary gel formation with xanthan

The influence of the galactomannan characteristic ratios (M/G) on the temperature of gelation (Tg) and the gel strength of mixtures of galactomannan with xanthan is reported. Two galactomannans were investigated: one highly substituted from the seeds of Mimosa scabrella (M/G = 11), and the other, less substituted, from the endosperm of Schizolobium parahybae, with (M/G = 30) [Ganter JLMS, Zawadzki-Baggio SF, Leitner SC, Sierakowski MR, Reicher F. J Carbohydr Chem 1993;12:753]. The xanthan:galactomannan systems (4:2 g l(-1), in 5 mM NaCl) showed a temperature of gel formation (Tg) of 24 degrees C for that of S. parahybae [Bresolin TMB, Milas M, Rinaudo M and Ganter JLMS. Int J Biol Macromol 1998;23:263] and 20 degrees C for the galactomannan of M. scabrella, determined by viscoelastic measurements and microcalorimetry. A Tg of 40-50 degrees C was found by Shatwell et al. [Shatwell KP, Sutherland IW, Ross-Murphy SB, Dea ICM. Carbohydr Polym 1991;14:29] for locust bean gum-LBG (M/G = 43). Lundin and Hermansson [Lundin L, Hermansson AM. Carbohydr Polym 1995;26:129] reported a difference of 13 degrees C for Tg of two LBG samples with M/G = 3 (40 degrees C) and 5 (53 degrees C), in mixtures with xanthan. It appears that the more substituted galactomannans have lower temperatures of gelation in the presence of xanthan. The mechanism of gelation depends also on the M/G ratio. For the lower values it involves only disordered xanthan chains in contrast to M/G ratios higher than 3. In addition, the presence of the galactomannan from M. scabrella increased slightly the temperature of the conformational change (Tm) of xanthan probably due to the ionic strength contribution of proteins (3.9%) present in the galactomannan. On the other hand, the galactomannans from S. parahybae, with 1.5% of proteins and M. scabrella, with 2.4% of protein, did not show this effect, the Tm of xanthan alone or in a mixture being practically unchanged.     

Potentiometric and electron nuclear double resonance properties of the two spin forms of the [4Fe-4S]+ cluster in the novel ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus.

Pyrococcus furiosus ferredoxin contains a single [4Fe-4S] that exists in both S = 1/2 (20%) and S = 3/2 (80%) ground states in the reduced protein. We report here on the temperature-dependent potentiometric properties of the two spin forms, their stability, and on the structural features that differentiate them. The midpoint potential (Em) of the cluster in either spin state was determined at -365 mV (30 degrees C, pH 8.0). By rapidly freezing samples for EPR analyses, it was shown that the Em values of both spin states appear to change by -1.7 mV/degrees C over the range 20 degrees-80 degrees C, and by -6 mV/degrees C between 80 and 89 degrees C. The Em values and the relative amounts of the S = 1/2 and S = 3/2 forms of the cluster were unaffected by pH (6.8-10.5), even at 85 degrees C, and were unchanged by the presence of NaCl (1.0 M), sodium dodecyl sulfate (10%, w/v) or ethylene glycol (50%, v/v), even at 80 degrees C. The S = 1/2 form of the [4Fe-4S]+ cluster was found to exhibit a strongly coupled 1H ENDOR resonance (A = 22 MHz) that was exchangeable with the solvent. Such a large coupling has not been observed in any other iron-sulfur protein. Since a unique feature of this 4Fe-ferredoxin is that only 3 cysteinyl residues appear to be coordinated to the [4Fe-4S] cluster, the ENDOR data are consistent with an H2O molecule being a ligand to the unique Fe site. The S = 3/2 form of the [4Fe-4S]+ cluster exhibited a similar, strongly coupled 1H ENDOR resonance, but in this spin state it was not exchangeable with the solvent. This suggests that the [4Fe-4S]+ cluster exhibiting the S = 3/2, but not the S = 1/2 ground state, is "shielded" from the solvent, presumably by neighboring amino acid residues. In view of the pH dependence of the midpoint potential of the two spin states, the fourth ligand to the cluster and the source of the strongly coupled 1H ENDOR resonance is probably an OH- rather than H2O molecule.     

The lactam of alpha-guanidinoglutaric acid (1-amidino-2-pyrrolidone-5-carboxylic acid)

alpha-Guanidinoglutaric acid (alpha-GGA) has been reported to occur in the cerebral cortex after epileptic seizures. No physical characteristics of alpha-GGA have been given. A practical procedure for the preparation of alpha-GGA is reported here. alpha-GGA forms a lactam in aqueous solution at 80 degrees C. It is proposed to substitute this lactam, 1-amidino-2-pyrrolidone-5-carboxylic acid (pAGlu), for pyroglutamic acid (pGlu) at the N-terminal position in neuropeptides to modify their biological characteristics. L(+)-Glutamic acid was reacted with S-methylisothiourea (I) at pH 10 in aqueous solution to form L(-)-alpha-guanidinoglutaric acid: mp 165-168 degrees C, [alpha]22D = -22.7 (C = 4, 2 M HCl). alpha-GGA reacted promptly with excess reagent to form a salt, S-methylisothiourea-alpha-guanidinoglutarate: mp 209-210 degrees C, [alpha]22D = -13.0 (C = 4, 2 M HCl). I was removed from the salt with aqueous picric acid, since I readily formed an insoluble picrate, S-methylisothiourea picrate (mp 225-228 degrees C). Alternatively, the salt was added to a cation exchange column, and the alpha-GGA was eluted with molar ammonium acetate buffer, pH 9.5. Its lactam, 1-amidino-2-pyrrolidone-5-carboxylic acid, mp 248-249 degrees C, [alpha]22D = +2.1 (C = 4, 2 M HCl), formed a picrate (mp 196-199 degrees C).