An orthorhombic crystal form of cyclohexaicosaose, CA26.32.59 H(2)O: comparison with the triclinic form.

Cycloamylose containing 26 glucose residues (cyclohexaicosaose, CA26) crystallized from water and 30% (v/v) polyethyleneglycol 400 in the orthorhombic space group P2(1)2(1)2(1) in the highly hydrated form CA26.32.59 H(2)O. X-ray analysis of the crystals at 0.85 A resolution shows that the macrocycle of CA26 is folded into two short left-handed V-amylose helices in antiparallel arrangement and related by a twofold rotational pseudosymmetry as reported recently for the (CA26)(2).76.75 H(2)O triclinic crystal form [Gessler, K. et al. Proc. Natl. Acad. Sci. USA 1999, 96, 4246-4251]. In the orthorhombic crystal form, CA26 molecules are packed in motifs reminiscent of V-amylose in hydrated and anhydrous forms. The intramolecular interface between the V-helices in CA26 is dictated by formation of an extended network of interhelical C-H...O hydrogen bonds; a comparable molecular arrangement is also evident for the intermolecular packing, suggesting that it is a characteristic feature of V-amylose interaction. The hydrophobic channels of CA26 are filled with disordered water molecules arranged in chains and held in position by multiple C-H...O hydrogen bonds. In the orthorhombic and triclinic crystal forms, the structures of CA26 molecules are equivalent but the positions of the individual water molecules are different, suggesting that the patterns of water chains are perturbed even by small structural changes associated with differences in packing arrangements in the two crystal lattices rather than with differences in the CA26 geometry.     

X-ray structure of the cyclomaltohexaicosaose triiodide inclusion complex provides a model for amylose-iodine at atomic resolution

Cyclomaltohexaicosaose (CA26) is folded into two 1(2)/(3) turns long V-helices that are oriented antiparallel. Crystals of complexes of CA26 with NH(4)I(3) and Ba(I(3))(2) are brown and X-ray analyses show that I(3)(-) units are located in the approximately 5 A wide central channels of the V-helices. In the complex with NH(4)I(3), two CA26 molecules are stacked to form 2 x 1(2)/(3) turns long channels harbouring 3 I(3)(-) at 3.66-3.85 A inter I(3)(-) distance (shorter than van der Waals distance, 4.3 A), whereas in the Ba(I(3))(2) complex, CA26 are not stacked and only one I(3)(-) each fills the V-helices. Glucose...I contacts are formed with C5-H, C3-H, C6-H and (at the ends of the V-helices) with O6 in (+) gauche orientation. By contrast, O2, O3, O4 and O6 in the preferred (-) gauche orientation do not interact with I because these distances are >/=4.01 A and exceed the van der Waals I...O sum of radii by about 0.5 A except for one O2...I distance of 3.68 A near the end of one V-helix. Raman spectra indicate that the complexes share the presence of I(3)(-) with blue amylose-iodine.     

A transgenic approach to understanding the influence of carbonic anhydrase on C18OO discrimination during C4 photosynthesis

The oxygen isotope composition of atmospheric CO(2) is an important signal that helps distinguish between ecosystem photosynthetic and respiratory processes. In C(4) plants the carbonic anhydrase (CA)-catalyzed interconversion of CO(2) and bicarbonate (HCO(3)(-)) is an essential first reaction for C(4) photosynthesis but also plays an important role in the CO(2)-H(2)O exchange of oxygen as it enhances the rate of isotopic equilibrium between CO(2) and water. The C(4) dicot Flaveria bidentis containing genetically reduced levels of leaf CA (CA(leaf)) has been used to test whether changing leaf CA activity influences online measurements of C(18)OO discrimination (Delta(18)O) and the proportion of CO(2) in isotopic equilibrium with leaf water at the site of oxygen exchange (theta). The Delta(18)O in wild-type F. bidentis, which contains high levels of CA relative to the rates of net CO(2) assimilation, was less than predicted by models of Delta(18)O. Additionally, Delta(18)O was sensitive to small decreases in CA(leaf). However, reduced CA activity in F. bidentis had little effect on net CO(2) assimilation, transpiration rates (E), and stomatal conductance (g(s)) until CA levels were less than 20% of wild type. The values of theta determined from measurements of Delta(18)O and the (18)O isotopic composition of leaf water at the site of evaporation (delta(e)) were low in the wild-type F. bidentis and decreased in transgenic plants with reduced levels of CA activity. Measured values of theta were always significantly lower than the values of theta predicted from in vitro CA activity and gas exchange. The data presented here indicates that CA content in a C(4) leaf may not represent the CA activity associated with the CO(2)-H(2)O oxygen exchange and therefore may not be a good predictor of theta during C(4) photosynthesis. Furthermore, uncertainties in the isotopic composition of water at the site of exchange may also limit the ability to accurately predict theta in C(4) plants.     

Metal-ion interactions with sugars. The crystal structure and FTIR study of an SrCl2-fructose complex

The single-crystal structure of SrCl2 x 2C6H12O6 x 3H2O was determined with Mr = 572.88, a = 16.252, b = 7.941(2), c = 10.751(3) angstroms, beta = 127.652(4) degrees, V = 1098.5(6) angstroms3, C2, Z = 2, mu = 0.71073 angstroms and R = 0.0296 for 1998 observed reflections. The fructose moiety of the complex exists as a beta-d-pyranose. The strontium atom is surrounded by eight oxygen atoms, which are arranged in symmetry-related pairs that are derived from four sugar and two water molecules. Three nonvicinal hydroxyl groups of fructose are involved in strontium binding. All the hydroxyl groups and water molecules are involved in forming an extensive hydrogen-bond network. The Sr-fructose complex is isostructural with the Ca-fructose complex, and the crystal structures and FTIR spectra of the two complexes are compared in this article. The O-H, C-O, and C-O-H vibrations are shifted, and the relative intensities changed in the complexes IR spectra, which indicate sugar metalation. By studying the metal-binding properties of fructose, it is hoped that such would aid in the understanding of the structural chemistry of metal ions interacting with saccharides, as an actual biological system, and thereby aid in the interpretation of some particular biological processes.     

Anti-oxidant activity of spermine and spermidine re-evaluated with oxidizing systems involving iron and copper ions

This study was designed to investigate the direction of redox reactions of spermine and spermidine in the presence of iron and copper. The redox activity of spermine and spermidine was assessed using a variety of methods, including their ability to: (1) reduce Fe(3+) to Fe(2+) ions; (2) protect deoxyribose from oxidation by Fe(2+)-ethylene diaminetetraacetic acid, Fe(3+)-ethylene diaminetetraacetic acid systems with and without H(2)O(2); (3) protect DNA from damage caused by Cu(2+)-H(2)O(2), and Fe(2+)-H(2)O(2) with and without ascorbic acid; (4) inhibit H(2)O(2)-peroxidase-induced luminol dependent chemiluminescence; (5) scavenge diphenyl-picryl-hydrazyl radical. Spermine and spermidine at concentration 1mM reduced 1.8+/-0.3 and 2.5+/-0.1 nmol of Fe(3+) ions during 20 min incubation. Both polyamines enhanced deoxyribose oxidation. The highest enhancement of 7.6-fold in deoxyribose degradation was found for combination of spermine with Fe(3+)-ethylene diaminetetraacetic acid. An 10mM spermine and spermidine decreased CuSO(4)-H(2)O(2)-ascorbic acid- and FeSO(4)-H(2)O(2)-ascorbic-induced DNA damage by 73+/-6, 69+/-4% and 90+/-5, 53+/-4%, respectively. They did not protect DNA from CuSO(4)-H(2)O(2) and FeSO(4)-H(2)O(2). Spermine apparently increased the CuSO(4)-H(2)O(2)-dependent injury to DNA. Polyamines attenuated H(2)O(2)-peroxidase-induced luminol dependent chemiluminescence. Total light emission from specimens containing 10mM spermine or spermidine was attenuated by 85.3+/-1.5 and 87+/-3.6%. During 20 min incubation 1mM spermine or spermidine decomposed 8.1+/-1.4 and 9.2+/-1.8% of diphenyl-picryl-hydrazyl radical. These results demonstrate that polyamines of well known anti-oxidant properties may act as pro-oxidants and enhance oxidative damage to DNA components in the presence of free iron ions and H(2)O(2).     

High-resolution mono- and multidimensional magic angle spinning 1H nuclear magnetic resonance of membrane peptides in nondeuterated lipid membranes and H2O.

High-speed (14 kHz) solid-state magic angle spinning (MAS) 1H NMR has been applied to several membrane peptides incorporated into nondeuterated dilauroyl or dimyristoylphosphatidylcholine membranes suspended in H2O. It is shown that solvent suppression methods derived from solution NMR, such as presaturation or jump-return, can be used to reduce water resonance, even at relatively high water content. In addition, regioselective excitation of 1H peptide resonances promotes an efficient suppression of lipid resonances, even in cases where these are initially two orders of magnitude more intense. As a consequence, 1H MAS spectra of the peptide low-field region are obtained without interference from water and lipid signals. These display resonances from amide and other exchangeable 1H as well as from aromatic nonexchangeable 1H. The spectral resolution depends on the specific types of resonance and membrane peptide. For small amphiphilic or hydrophobic oligopeptides, resolution of most individual amide resonance is achieved, whereas for the transmembrane peptide gramicidin A, an unresolved amide spectrum is obtained. Partial resolution of aromatic 1H occurs in all cases. Multidimensional 1H-MAS spectra of membrane peptides can also be obtained by using water suppression and regioselective excitation. For gramicidin A, F2-regioselective 2D nuclear Overhauser effect spectroscopy (NOESY) spectra are dominated by intermolecular through-space connectivities between peptide aromatic or formyl 1H and lipid 1H. These appear to be compatible with the known structure and topography of the gramicidin pore. On the other hand, for the amphiphilic peptide leucine-enkephalin, F2-regioselective NOESY spectra mostly display cross-peaks originating from though-space proximities of amide or aromatic 1H with themselves and with aliphatic 1H. F3-regioselective 3D NOESY-NOESY spectra can be used to obtain through-space correlations within aliphatic 1H. Such intrapeptide proximities should allow determination of the conformation of the peptide in membranes. It is suggested that high-speed MAS multidimensional 1H NMR of peptides in nondeuterated membranes and in H2O can be used for studies of both peptide structure and lipid-peptide interactions.     

Comparative substrate recognition by bacterial and fungal purine transporters of the NAT/NCS2 family

We compared the interactions of purines and purine analogues with representative fungal and bacterial members of the widespread Nucleobase-Ascorbate Transporter (NAT) family. These are: UapA, a well-studied xanthine-uric acid transporter of A. nidulans, Xut1, a novel transporter from C. albicans, described for the first time in this work, and YgfO, a recently characterized xanthine transporter from E. coli. Using transport inhibition experiments with 64 different purines and purine-related analogues, we describe a kinetic approach to build models on how NAT proteins interact with their substrates. UapA, Xut1 and YgfO appear to bind several substrates via interactions with both the pyrimidine and imidazol rings. Fungal homologues interact with the pyrimidine ring of xanthine and xanthine analogues via H-bonds, principally with N1-H and =O6, and to a lower extent with =O2. The E. coli homologue interacts principally with N3-H and =O2, and less strongly with N1-H and =O6. The basic interaction with the imidazol ring appears to be via a H-bond with N9. Interestingly, while all three homologues recognize xanthines with similar high affinities, interaction with uric acid or/and oxypurinol is transporter-specific. UapA recognizes uric acid with high affinity, principally via three H-bonds with =O2, =O6 and =O8. Xut1 has a 13-fold reduced affinity for uric acid, based on a different set of interactions involving =O8, and probably H atoms from positions N1, N3, N7 or N9. YgfO does not recognize uric acid at all. Both Xut1 and UapA recognize oxypurinol, but use different interactions reflected in a nearly 26-fold difference in their affinities for this drug, while YgfO interacts with this analogue very inefficiently.     

Crystallization and X-ray crystallographic studies of wild-type and mutant tryptophan synthase alpha-subunits from Escherichia coli

The alpha-subunit of Escherichia coli tryptophan synthase (aTS), a component of the tryptophan synthase alpha2beta2 complex, is a monomeric 268-residues protein (Mr = 28,600). alphaTS by itself catalyzes the cleavage of indole-3-glycerol phosphate to glyceraldehyde-3-phosphate and indole, which is converted to tryptophan in tryptophan biosynthesis. Wild-type and P28L/Y173F double mutant alpha-subunits were overexpressed in E. coli and crystallized at 298 K by the hanging-drop vapor-diffusion method. X-ray diffraction data were collected to 2.5 angstroms resolution from the wild-type crystals and to 1.8 angstroms from the crystals of the double mutant, since the latter produced better quality diffraction data. The wild-type crystals belonged to the monoclinic space group C2 (a = 155.64 angstroms, b = 44.54 angstroms, c = 71.53 angstroms and beta = 96.39 degrees) and the P28L/Y173F crystals to the monoclinic space group P21 (a = 71.09 angstroms, b = 52.70, c = 71.52 angstroms, and beta = 91.49 degrees). The asymmetric unit of both structures contained two molecules of aTS. Crystal volume per protein mass (V(m)) and solvent content were 2.15 angstroms3 Da(-1) and 42.95% for the wild-type and 2.34 angstroms3 Da(-1) and 47.52% for the double mutant.     

Structure of a neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B26

The neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B26 in skimmed milk was found to be composed of d-glucose and d-galactose in a molar ratio of 2:3. Linkage analysis and 1D/2D NMR ((1)H and (13)C) studies performed on the native polysaccharide, and on an oligosaccharide obtained from a partial acid hydrolysate of the native polysaccharide, showed the polysaccharide to consist of branched pentasaccharide repeating units with the following structure. [structure: see text]     

NM23-H1 tumor suppressor physically interacts with serine-threonine kinase receptor-associated protein, a transforming growth factor-beta (TGF-beta) receptor-interacting protein, and negatively regulates TGF-beta signaling

NM23-H1 is a member of the NM23/NDP kinase gene family and a putative metastasis suppressor. Previously, a screen for NM23-H1-interacting proteins that could potentially modulate its activity identified serine-threonine kinase receptor-associated protein (STRAP), a transforming growth factor (TGF)-beta receptor-interacting protein. Through the use of cysteine to serine amino acid substitution mutants of NM23-H1 (C4S, C109S, and C145S) and STRAP (C152S, C270S, and C152S/C270S), we demonstrated that the association between these two proteins is dependent on Cys(145) of NM23-H1 and Cys(152) and Cys(270) of STRAP but did not appear to involve Cys(4) and Cys(109) of NM23-H1, suggesting that a disulfide linkage involving Cys(145) of NM23-H1 and Cys(152) or Cys(270) of STRAP mediates complex formation. The interaction was dependent on the presence of dithiothreitol or beta-mercaptoethanol but not H(2)O(2). Ectopic expression of wild-type NM23-H1, but not NM23-H1(C145S), negatively regulated TGF-beta signaling in a dose-dependent manner, enhanced stable association between the TGF-beta receptor and Smad7, and prevented nuclear translocation of Smad3. Similarly, wild-type NM23-H1 inhibited TGF-beta-induced apoptosis and growth inhibition, whereas NM23-H1(C145S) had no effect. Knockdown of NM23-H1 by small interfering RNA stimulated TGF-beta signaling. Coexpression of wild-type STRAP, but not STRAP(C152S/C270S), significantly stimulated NM23-H1-induced growth of HaCaT cells. These results suggest that the direct interaction of NM23-H1 and STRAP is important for the regulation of TGF-beta-dependent biological activity as well as NM23-H1 activity.     

Isolation and structural identification of a direct-acting mutagen derived from N-nitroso-N-methylpentylamine and Fenton's reagent with copper ion

N-Nitrosodialkylamines show their mutagenicity by forming α-hydroxynitrosamines in the presence of rat S9 mix in the Ames assay. The hydroxyl radical derived from Fe(2+)-H(2)O(2) (Fenton's reagent) with Cu(2+) activates N-nitrosamines, with an alkyl chain longer than a propyl constituent, to a direct-acting mutagen. The reactivity of Fe(2+)-Cu(2+)-H(2)O(2) on nitrosamines in relation to their metabolic activation is not fully characterized. Here, we report the identification of the direct-acting mutagen derived from N-nitroso-N-methylpentylamine (NMPe) in the presence of Fe(2+), Cu(2+), H(2)O(2) and nitric oxide (NO), which is a product of nitrosamine metabolism. A dichloromethane extract of the NMPe reaction mixtures was fractionated by silica gel column chromatography several times and by a preparative high performance liquid chromatography (HPLC); we obtained white crystals as a product. The direct-acting mutagen that was isolated was provisionally identified as 5-ethyl-5-nitro-1-pyrazoline 1-oxide by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy and X-ray crystallography. To confirm the structure of the mutagen, the authentic compound was synthesized from 2-nitrobutene and diazomethane, followed by N-oxidation with m-chloroperoxybenzoic acid. The (1)H NMR spectral data from the direct-acting mutagen that was synthesized was identical to the data from the isolated mutagen. Furthermore, the authentic 5-ethyl-5-nitro-1-pyrazoline 1-oxide was mutagenic in Salmonella typhimurium TA1535. The results showed that 5-ethyl-5-nitro-1-pyrazoline 1-oxide was a direct-acting mutagen derived from the reaction of NMPe and Fe(2+)-Cu(2+)-H(2)O(2)-NO.     

An investigation of the inclusion complex of beta-cyclodextrin with p-nitrobenzoic acid in the solid state

The weak inclusion complex of cyclomaltoheptaose (beta-cyclodextrin, betaCD) with p-nitrobenzoic acid was investigated in the solid state. Crystallography shows that two betaCD molecules co-crystallize with two p-nitrobenzoic acids and 28.5 water molecules [2(C(42)H(70)O(35))x2(C(7)H(5)NO(4))x28.5H(2)O] in the triclinic system.     

Comparative substrate recognition by bacterial and fungal purine transporters of the NAT/NCS2 family

We compared the interactions of purines and purine analogues with representative fungal and bacterial members of the widespread Nucleobase-Ascorbate Transporter (NAT) family. These are: UapA, a well-studied xanthine-uric acid transporter of A. nidulans, Xut1, a novel transporter from C. albicans, described for the first time in this work, and YgfO, a recently characterized xanthine transporter from E. coli. Using transport inhibition experiments with 64 different purines and purine-related analogues, we describe a kinetic approach to build models on how NAT proteins interact with their substrates. UapA, Xut1 and YgfO appear to bind several substrates via interactions with both the pyrimidine and imidazol rings. Fungal homologues interact with the pyrimidine ring of xanthine and xanthine analogues via H-bonds, principally with N1-H and =O6, and to a lower extent with =O2. The E. coli homologue interacts principally with N3-H and =O2, and less strongly with N1-H and =O6. The basic interaction with the imidazol ring appears to be via a H-bond with N9. Interestingly, while all three homologues recognize xanthines with similar high affinities, interaction with uric acid or/and oxypurinol is transporter-specific. UapA recognizes uric acid with high affinity, principally via three H-bonds with =O2, =O6 and =O8. Xut1 has a 13-fold reduced affinity for uric acid, based on a different set of interactions involving =O8, and probably H atoms from positions N1, N3, N7 or N9. YgfO does not recognize uric acid at all. Both Xut1 and UapA recognize oxypurinol, but use different interactions reflected in a nearly 26-fold difference in their affinities for this drug, while YgfO interacts with this analogue very inefficiently.     

Macrophage activation and histopathological findings in Calomys callosus and Swiss mice infected with several strains of Trypanosoma cruzi.

Peritoneal macrophage activation as measured by H2O2 release and histopathology was compared between Swiss mice and Calomys callosus, a wild rodent, reservoir of Trypanosoma cruzi, during the course of infection with four strains of this parasite. In mice F and Y strain infections result in high parasitemia and mortality while with silvatic strains Costalimai and M226 parasitemia is sub-patent, with very low mortality. H2O2 release peaked at 33.6 and 59 nM/2 x 10(6) cells for strains Y and F, respectively, 48 and 50 nM/2 x 10(6) for strains Costalimai and M226, at different days after infection. Histopathological findings of myositis, myocarditis, necrotizing arteritis and absence of macrophage parasitism were found for strains F and Costalimai. Y strain infection presented moderate myocarditis and myositis, with parasites multiplying within macrophages. In C. callosus all four strains resulted in patent parasitemia which was eventually overcome, with scarce mortality. H2O2 release for strains Y and F was comparable to that of mice-peaks of 27 and 53 nM/2 x 10(6) cells, with lower values for strains Costalimai and M226-16.5 and 4.6 nM/2 x 10(6) cells, respectively. Histopathological lesions with Y and F strain injected animals were comparable to those of mice at the onset of infections; they subsided completely at the later stages with Y strain and partially with F strain infected C. callosus. In Costalimai infected C. callosus practically no histopathological alterations were observed.     

Two γ-Methyl Biosides from Dregea volubilis (L.) Benth

From the hydrolysate of the crude glycosides from the roots, of Dregea volubilis(L.) Benth in Dehong, Yunnan, two α-methyl biosides Ⅰ and Ⅱ (yields: 0.016%and 0.0097%, respectively) were isolated by silica gel column chromatography. Theirchemical structures were established by interpretation of MS, IR,1H,13C-NMR, andgas chromatographic analysis of their degradation products, and comparison of thephysical properties of Ⅰ, Ⅱ and their acetates which were reported in literatures asfollows: α-methyl-pachybioside for Ⅰ, and α-methyl-[3-O-methyl-6-deoxy-D-allose(1→4)-D-olivoside] for Ⅱ. Ⅱ named α-methyl-dredehongbioside, is reported for the first time. Ⅱ, α-methyl-dredehongbioside, colorless needles (from MeOH), bitter, mp. 184–186℃,[α]D22 +74.5˚~(c= 0.52, MeOH). Anal. Cald(%) for C14H26O8:C52.17, H8.07;Found; C52.23,H8.22. Irvmaxkbr: 3370, 1443, 1419, 1375, 1268, 1218, 1168,1127,1060cm-1. MS(m/e,%): 322(M+,3),291(M+-OCH3,15), 273(M+-OCH3-H2O,12), 258,246,232, 222, 159, 145, 141, 128, 95, 87, 85, 74 (base peak, 100), 59. 1H NMRδ(CDCl3): 4.73(1H, dd, J= 4.0 Hz, J= 1.5Hz, C-1-H), 4.55(1H, d, J= 8.0Hz,C-1′-H), 3.79(1H, dd, J=3.0Hz, J= 3.0Hz, C-3′-H), 3.00(1H, dd, J= 9.0Hz,J= 9.0Hz, C-4-H), 2.22(1H, m, C-2-Ha), 1.60(1H, m, C-2-He), 1.33(3H, d,J= 6.0Hz,C-5-CH,), 1.31(3H,d,,J= 6.5Hz, C-5′-CH), 3.68(3H,s,,C-3′-OCH),3.31(3H,s,C-1-OCH). 13C NMR data were seen in Table 1. Ⅳ, tri-acetyl-α-methyl-dredehongbioside, colorless granular (from MeOH),mp. 135--137℃, [a]D22+ 88.2˚(c= 0.50, MeOH). MS(m/e, %): 488 (M+, 2), 388(M+-HOAc,2), 357(M+-OCH3-HOAc,33), 288, 187, 127, 116, 85, 74, 59, 43(basepeak, 100). 1H NMR,δ(CDCl3): 5.25(1H, ddd,.J= 11.0Hz, J=9.0Hz,.J= 5.5Hz,C-3-H), 4.86(1H, d, J= 8.0Hz, C-1′-H), 4.69 (1H, dd, J= 4.0Hz, J= 1.5Hz,C-1-H), 4.58(1H,m,C-2′-H),3.94(1H, dd, J= 3.0Hz,J= 3.0Hz,C-3′-H),3.64(1H,m,C-5-H), 3.22(1H,dd, J= 9.5Hz, J= 8.5Hz, C-4-H), 2.30(1H, m, C-2-Ha),1.67(1H,m,C-2-He), 1.31(3H, d, .J= 6.5Hz, C-5-CH3), 1.17(3H, d, J= 6.0Hz,C-5-CH3), 3.47(3H, s, C-3′-OCH3), 3.30(3H,s, C-1-OCH3), 2.10(6H, s, C-2′, C- 4′ -OCH3), 2.03(3H,s,C-3-OCH3).     

Synthesis, crystal structure, cytotoxic activity and DNA-binding properties of the copper (II) and zinc (II) complexes with 1-[3-(2-pyridyl)pyrazol-1-ylmethyl]naphthalene

A new ligand L, 1-[3-(2-pyridyl)pyrazol-1-ylmethyl]naphthalene, and its two metal complexes, [Cu(L)3](ClO4)2 (1) and [Zn(L)3](ClO4)2(H2O)2 (2), have been synthesized and characterized. The crystal structure of complex 1 was determined by single crystal X-ray diffraction, which crystallized in monoclinic, space group P2(1)/n with unit cell parameters, a = 12.710(4) angstroms, b = 12.135(3) angstroms, c = 33.450(9) angstroms, beta = 93.281(5) degrees and Z = 4. The Cu atom was six-coordinated to N(1), N(2), N(4), N(5), N(7) and N(8) from three L ligands and formed a slightly distorted octahedral geometry. Complexes 1 and 2, and ligand L were subjected to biological tests in vitro using three different cancer cell lines (HL-60, BGC-823 and MDA-MB-435). Complex 1 showed significant cytotoxic activity against three cancer cell lines. The interactions of complexes 1 and 2, and ligand L with calf thymus DNA were then investigated by thermal denaturation, viscosity measurements and spectrophotometric methods. The experimental results indicated that complexes 1 and 2 bound to DNA by intercalative mode via the ligand L. The intrinsic binding constants of complexes 1 and 2, and ligand L with DNA were 1.8 x 10(4), 5.4 x 10(3) and 2.76 x 10(3) M(-1), respectively.     

Two-dimensional NMR studies of staphylococcal nuclease: evidence for conformational heterogeneity from hydrogen-1, carbon-13, and nitrogen-15 spin system assignments of the aromatic amino acids in the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex

A combination of multinuclear two-dimensional NMR experiments served to identify and assign the combined 1H, 13C, and 15N spin systems of the single tryptophan, three phenylalanines, three histidines, and seven tyrosines of staphylococcal nuclease H124L in its ternary complex with calcium and thymidine 3',5'-bisphosphate at pH 5.1 (H2O) or pH 5.5 (2H2O). Samples of recombinant nuclease were labeled with 13C or 15N as appropriate to individual NMR experiments: uniformly with 15N (all sites to greater than 95%), uniformly with 13C (all sites to 26%), selectively with 13C (single amino acids uniformly labeled to 26%), or selectively with 15N (single amino acids uniformly labeled to greater than 95%). NMR data used in the analysis included single-bond and multiple-bond 1H-13C and multiple-bond 1H-15N correlations, 1H-13C single-bond correlation with Hartmann-Hahn relay (1H[13C]SBC-HH), and 1H-13C single-bond correlation with NOE relay (1H[13C]SBC-NOE). The aromatic protons of the spin systems were identified from 1H[13C]SBC-HH data, and the nonprotonated aromatic ring carbons were identified from 1H-13C multiple-bond correlations. Sequence-specific assignments were made on the basis of observed NOE relay connectivities between assigned 1H alpha-13C alpha or 1H beta-13C beta direct cross peaks in the aliphatic region [Wang, J., LeMaster, D. M., & Markley, J. L. (1990) Biochemistry 29, 88-101] and 1H delta-13C delta direct cross peaks in the aromatic region of the 1H[13C]SBC-NOE spectrum. The His121 1H delta 2 resonance, which has an unusual upfield shift (at 4.3 ppm in the aliphatic region), was assigned from 1H[13C]SBC, 1H[13C]MBC, and 1H[15N]MBC data. Evidence for local structural heterogeneity in the ternary complex was provided by doubled peaks assigned to His46, one tyrosine, and one phenylalanine. Measurement of NOE buildup rates between protons on different aromatic residues of the major ternary complex species yielded a number of interproton distances that could be compared with those from X-ray structures of the wild-type nuclease ternary complex with calcium and thymidine 3',5'-bisphosphate [Cotton, F. A., Hazen, E. E., Jr., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555; Loll, P. J., & Lattman, E. E. (1989) Proteins: Struct., Funct., Genet. 5, 183-201]. The unusual chemical shift of His121 1H delta 2 is consistent with ring current calculations from either X-ray structure.     

8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 2. Kinetic and hydrogen-transfer studies

Steady-state kinetic parameters have been obtained for the pure 8-hydroxy-5-deazaflavin-reducing hydrogenase. With H2 and 8-hydroxy-5-deazariboflavin (F0) as substrates, Km (H2) = 12 microM, Km (F0) = 26 microM, and Kcat = 225 s-1. In the back-direction, F0H2 is reoxidized (anaerobically) at 225 s-1. Initial velocity patterns, product inhibition patterns, dead-end inhibition by carbon monoxide, and transhydrogenation to Procion Red HE-3B suggest a two-site hybrid ping-pong mechanism. A kinetic derivation for the rate equation is provided in the Appendix. Studies with D2 and with D2O reveal that no steps involving D transfer are substantially rate determining. Further, D2 yields F0H2 with no deuterium at C5 while in D2O a 5-monodeuterio F0H2 product is formed, indicating complete exchange of hydrogens from H2 with solvent before final transfer of a hydride ion out from reduced enzyme to C5 of F0.     

Insertion mutagenesis of wca reduces acid and heat tolerance of enterohemorrhagic Escherichia coli O157:H7

Strains of enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 produce under stress copious amounts of exopolysaccharide (EPS) composed of colanic acid (CA). Studies were performed to evaluate the association of production of CA with survival of EHEC under adverse environmental conditions. A CA-deficient mutant, M4020, was obtained from a CA-proficient parental strain, E. coli O157:H7 W6-13, by inserting a kanamycin resistance gene cassette (kan) into wcaD and wcaE, 2 of the 21 genes required for CA biosynthesis. M4020 was defective in CA production as determined from the ratio of uronic acid to protein (UA/P) of cells grown from 1 to 4 days at 25 degrees C on minimal glucose agar (MGA), MacConkey agar, and sorbitol-MacConkey agar, and by colony morphology on MGA. The results of stress treatment revealed that M4020 was substantially less tolerant to acid (pH 4.5 and 5.5) and heat (55 and 60 degrees C) in comparison to W6-13, indicating that CA confers on E. coli O157:H7 a protective effect from the environmental stresses of acid and heat.     

Copper(II) complexes of salicylaldehyde hydrazones: synthesis, structure, and DNA interaction

Three hydrazone ligands, H2L1-H2L3, made from salicylaldehyde and ibuprofen- or naproxen-derived hydrazides, were prepared and transformed into the corresponding copper(II) complexes [Cu(II)L1] x H2O, [Cu(II)L2], and [(Cu(II))2(L3)2] x H2O x DMF (Scheme). The X-ray crystal structure of the last-mentioned complex was solved (Fig. 1), showing a square-planar complexation geometry, and the single units were found to form a one-dimensional chain structure (Fig. 2). The interactions of these complexes with CT-DNA were studied by different techniques, indicating that they all bind to DNA by classical and/or non-classical intercalation modes.