Hydrogen bonding in thiolbenzoic acid. Infra-red, ultra-violet, n.m.r. and cryoscopic studies

Dimerization of thiolbenzoic acid has been studied by infra-red, ultra-violet and n.m.r. spectroscopy and cryoscopy. The results indicate that the tendency to form S - H. O hydrogen bonds is not appreciable.

Molecular structure of Lantadene-B&C, triterpenoids ofantana camara, red variety: Lantadene-B, 22β-angeloyloxy-3-oxoolean-12-en-28-oic acid; Lantadene-C, 22 β-(S)-2′-methyl butanoyloxy-3-oxoolean-12-en-28-oic acid

(I)Lantadene-B: C35H52O5,M r =552.80, MonoclinicC2,a=25.65(1),b=6.819(9),c=18.75(1) Å,beta=100.61(9),V=3223(5) Å3,Z=4,D x =1.14 g cm–3 CuKagr (lambda=1.5418A),mgr=5.5 cm–1,F(000)=1208,R=0.118,wR=0.132 for 1527 observed reflections withF o ge2sgr(F o ). (II)Lantadene-C: C35H54O5·CH3OH,Mr=586.85, Monoclinic,P21,a=9.822(3),b=10.909(3),c=16.120(8)Å,beta=99.82(4),V=1702(1)Å3,Z=2,D x =1.145 g cm–3, MoKagr (lambda=0.7107Å), mgr=0.708 cm–1 F(000)=644,R=0.098, wR=0.094 for 1073 observed reflections. The rings A, B, C, D, and E aretrans, trans, trans, cis fused and are in chair, chair, sofa, half-chair, chair conformations, respectively, in both the structures. In the unit cell the molecules are stabilized by O-HctdotO hydrogen bonds in both the structures, however an additional C-HctdotO interaction is observed in the case of Lantadene-C.

Purification of 3,5-Dichlorocatechol 1,2-Dioxygenase, a Nonheme Iron Dioxygenase and a Key Enzyme in the Biodegradation of a Herbicide, 2,4-Dichlorophenoxyacetic acid (2,4-D), from Pseudomonas cepacia CSV90

An enzyme which cleaves the benzene ring of 3,5-dichiorocatechol has been purified to homogeneity from Pseudomonas cepacia CSV90, grown with 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon source. The enzyme was a nonheme ferric dioxygenase and catalyzed the intradiol cleavage of all the examined catechol derivatives, 3,5-dichlorocatechol having the highest specificity constant of 7.3 μM−1 s−1 in an air-saturated buffer. No extradiol-cleaving activity was observed. Thus, the enzyme was designated as 3,5-dichlorocatechol 1,2-dioxygenase. The molecular weight of the native enzyme was ascertained to be 56,000 by light scattering method, while the Mr value of the enzyme denatured with 6 M guanidine-HCl or sodium dodecyl sulfate was 29,000 or 31,600, respectively, suggesting that the enzyme was a homodimer. The iron content was estimated to be 0.89 mol per mole of enzyme. The enzyme was deep red and exhibited a broad absorption spectrum with a maximum at around 425 nm, which was bleached by sodium dithionite, and shifted to 515 nm upon anaerobic 3,5-dichlorocatechol binding. The catalytic constant and the Km values for 3,5-dichlorocatechol and oxygen were 34.7 s−1 and 4.4 and 652 μM, respectively, at pH 8 and 25°C. Some heavy metal ions, chelating agents and sulfhydryl reagents inhibited the activity. The NH2-terminal sequence was determined up to 44 amino acid residues and compared with those of the other catechol dioxygenases previously reported.

Susceptibility of sperm chromatin to acid denaturation in situ: A study in endogenous FSH-deprived adult male bonnet monkeys (Macaca radiata)

Acid denaturation of calf thymus DNA in vitro followed by acridine orange (AO) binding induced a 112% increase in the emission of red, a 58% decrease in green, and a consequential decrease in the ratio of green:red fluorescences from 1.7 to 0.9. This metachromatic property of AO on binding to DNA following acid denaturation was utilized to study the susceptibility of normal and ovine follicle-stimulating hormone (oFSH) actively immunized bonnet monkey spermatozoa voided throughout the year. For analyses, the scattergram generated by the emission of red and green fluorescences by 10,000 AO-bound sperm from each semen sample was divided into 4 quadrant zones representing percentage cells fluorescing high green-low red (Q1), high green-high red (Q2), low green-low red (Q3) and low green-high red. (Q4). Normal monkey sperm obtained during the months of July-December exhibited 76, 13, and 11% cells in Q2, Q3, and Q4 quadrants, respectively. However, during January-June, when the females of the species are markedly subfertile, noncycling, and amenorrhoeic, the spermatozoa ejaculated by the male monkeys exhibited 38, 39, and 23% sperm in Q2, Q3, and Q4, respectively, the differences being highly significant (p < .01-.001). FSH deprivation induced significant shifts in fluorescence emissions, from respective controls, with 39, 33, and 28% cells in Q2, Q3, and Q4, respectively, during July-December, and 15, 48, and 37% sperm in Q2, Q3, and Q4 quadrants, respectively, during January-June. It is postulated that the altered kinetics of germ cell transformations and the deficient spermiogenesis observed earlier following FSH deprivation in these monkeys may have induced the enhanced susceptibility to acid denaturation in sperm.

Heme biosynthesis by the malarial parasite - Import of delta-aminolevulinate dehydrase from the host red cell

The mouse and human malarial parasites, Plasmodium berghei and Plasmodium falciparum, respectively, synthesize heme de novo following the standard pathway observed in animals despite the availability of large amounts of heme, derived from red cell hemoglobin, which is stored as hemozoin pigment, The enzymes, delta-aminolevulinate dehydrase (ALAD), coproporphyrinogen oxidase, and ferrochelatase are present at strikingly high levels in the P, berghei infected mouse red cell in vivo, The isolated parasite has low levels of ALAD and the data clearly indicate it to be of red cell origin. The purified enzyme preparations from the uninfected red cell and the parasite are identical in kinetic properties, subunit molecular weight, cross-reaction with antibodies to the human enzyme, and N-terminal amino acid sequence. Immunogold electron microscopy of the infected culture indicates that the enzyme is present inside the parasite and, therefore, is not a contaminant, The parasite derives functional ALAD from the host and the enzyme binds specifically to isolated parasite membrane in vitro, suggestive of the involvement of a receptor in its translocation into the parasite, While, ALAD, coproporphyrinogen oxidase, and ferrochelatase from the parasite and the uninfected red cell supernatant have identical subunit molecular weights on SDS-polyacrylamide gel electrophoresis and show immunological cross-reaction with antibodies to the human enzymes, as revealed by Western analysis, the first enzyme of the pathway, namely, delta-aminolevulinate synthase (ALAS) in the parasite, unlike that of the red cell host, does not cross-react with antibodies to the human enzyme, However, ALAS enzyme activity in the parasite is higher than that of the infected red cell supernatant. We therefore conclude that the parasite, while making its own ALAS, imports ALAD and perhaps most of the other enzymes of the pathway from the host to synthesize heme de novo, and this would enable it to segregate this heme from the heme derived from red cell hemoglobin degradation, ALAS of the parasite and the receptor(s) involved in the translocation of the host enzymes into the parasite would be unique drug targets.

Copper(II) Complexes of l-Arginine as Netropsin Mimics Showing DNA Cleavage Activity in Red Light

Copper(II) complexes [Cu(L-arg)(2)](NO3)(2) (1) and [Cu(L-arg)(B)Cl]Cl (2-5), where B is a heterocyclic base, namely, 2,2'-bipyridine (bpy, 2), 1,10-phenanthroline (phen, 3), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 4), and dipyrido[3,2-a:2',3'-c)phenazine (dppz, 5), are prepared and their DNA binding and photoinduced DNA cleavage activity studied. Ternary complex 3, structurally characterized using X-ray crystallography, shows a square-pyramidal (4 + 1) coordination geometry in which the N,O-donor L-arginine and N,N-donor 1,10-phenanthroline form the basal plane with one chloride at the elongated axial site. The complex has a pendant cationic guanidinium moiety. The one-electron paramagnetic complexes display a metal-centered d-d band in the range of 590-690 nm in aqueous DMF They show quasireversible cyclic voltammetric response due to the Cu(II)/Cu(I) couple in the range of -0.1 to -0.3 V versus a saturated calomel electrode in a DMF-Tris HCl buffer (pH 7.2). The DNA binding propensity of the complexes is studied using various techniques. Copper(II) bis-arginate 1 mimics the minor groove binder netropsin by showing preferential binding to the AT-rich sequence of double-strand (ds) DNA. DNA binding study using calf thymus DNA gives an order: 5 (L-arg-dppz) >= 1 (biS-L-arg) > 4 (L-arg-dpq) > 3 (L-arg-phen) >> 2 (L-arg-bpy). Molecular docking calculations reveal that the complexes bind through extensive hydrogen bonding and electrostatic interactions with ds-DNA. The complexes cleave supercoiled pUC19 DNA in the presence of 3-mercaptopropionic acid as a reducing agent forming hydroxyl ((OH)-O-center dot) radicals. The complexes show oxidative photoinduced DNA cleavage activity in UV-A light of 365 nm and red light of 647.1 nm (Ar-Kr mixed-gas-ion laser) in a metal-assisted photoexcitation process forming singlet oxygen (O-1(2)) species in a type-II pathway. All of the complexes, barring complex 2, show efficient DNA photocleavage activity. Complexes 4 and 5 exhibit significant double-strand breaks of DNA in red light of 647.1 nm due to the presence of two photosensitizers, namely, L-arginine and dpq or dppz in the molecules.

A thermal molecular migration in the solid-state - structures of isomeric 5-amino-4-(2,6-dichlorophenyl)-1-(2-nitrophenyl)-1h-1,2,3-triazole (yellow form i) and 4-(2,6-dichlorophenyl)-5-(2-nitroanilino)-2h-1,2,3-triazole (red form-ii), c14h9cl2n5o2

Yellow form (I): Mr= 350.09, monoclinic, P2Jn, Z--4, a=9.525(1), b=14.762(1), c= 11.268(1),/t, fl= 107.82 (1) o , V= 1508.3 A 3 , Din(flotation in aqueous KI)= 1.539 (2), D x= 1.541 (2) g cm -3, #(Cu Ka, 2 = 1.5418 A) = 40.58 cm -~, F(000) = 712, T= 293 K, R = 8.8% for 2054 significant refections. Red form (II): Mr= 350.09, triclinic, Pi, Z=2, a=9.796(2), b= 10.750 (2), c= 7.421 (1)A, a= 95.29 (2), fl= 0108-2701/84/111901-05501.50 70.18 (1), y = 92-.76 (2) °, V= 731.9 A 3, Din(flotation in KI) = 1.585 (3), D x = 1.588 (3) g cm -3, ~t(Cu Ka, 2 = 1.5418/~) = 40.58 cm -1, F(000) = 356, T=293 K, R = 5.8% for 1866 significant reflections. There are no unusual bond distances or angles. The triazole and two phenyl rings are planar. On the basis of packing considerations the possibility of intermolecular interactions playing a role in the reactivity of the starting material is ruled out.

Enzyme-catalysed non-oxidative decarboxylation of aromatic acids: I.Purification and spectroscopic properties of 2,3 dihydroxybenzoic acid decarboxylase from Image Image

In order to understand the molecular mechanism of non-oxidative decarboxylation of aromatic acids observed in microbial systems, 2,3 dihydroxybenzoic acid (DHBA) decarboxylase from Image Image was purified to homogeneity by affinity chromatography. The enzyme (Mr 120 kDa) had four identical subunits (28 kDa each) and was specific for DHBA. It had a pH optimum of 5.2 and Km was 0.34mM. The decarboxylation did not require any cofactors, nor did the enzyme had any pyruvoyl group at the active site. The carboxyl group and hydroxyl group in the Image -position were required for activity. The preliminary spectroscopic properties of the enzyme are also reported.

Peptide models for the bacteriorhodopsin chromophore. Retinylidene-lysine systems containing aspartic acid and serine residues

The retinylidene Schiff base derivative of seven lysine containing peptides have been prepared in order to investigate solvent and neighboring group effects, on the absorption maximum of the protonated Schiff base chromophore. The peptides studied are Boc-Aib-Lys-Aib-OMe (1), Boc-Ala-Aib-Lys-OMe (2), Boc-Ala-Aib-Lys-Aib-OMe (3), Boc-Aib-Asp-Aib-Aib-Lys-Aib-OMe (4), Boc-Aib-Asp-Aib-Ala-Aib-Lys-Aib-OMe (5), Boc-Lys-Val-Gly-Phe-OMe (6) and Boc-Ser-Ala-Lys-Val-Gly-Phe-OMe (7). In all cases protonation shifts the absorption maxima to the red by 3150–8450 cm-1. For peptides 1–3 the protonation shifts are significantly larger in nonhydrogen bonding solvents like CHCl3 or CH2Cl2 as compared to hydrogen bonding solvents like CH3OH. The presence of a proximal Asp residue in 4 and 5 results in pronounced blue shift of the absorption maximum of the protonated Schiff base in CHCl3, relative to peptides lacking this residue. Peptides 6 and 7 represent small segments of the bacteriorhodopsin sequence in the vicinity of Lys-216. The presence of Ser reduces the magnitude of the protonation shift.

Involvement of protocatechuic acid in the metabolism of phenylacetic acid by Aspergillus niger

Abstract is not available.

Photocytotoxic 3d-Metal Scorpionates with a 1,8-Naphthalimide Chromophore Showing Photoinduced DNA and Protein Cleavage Activity

Ternary 3d-metal complexes of formulation [M(Tp(Ph))(py-nap)](ClO4)(1-3), where M is Co(II) (1), Cu(II) (2), and Zn(II) (3); Tp(Ph) is anionic tris (3-phenylpyrazolyl)borate; and py-nap is a pyridyl ligand with a conjugated 1,8-naphthalimide moiety, have been prepared from the reaction of metal perchlorate with KTp(Ph) and py-nap in CH2Cl2. The complexes have been characterized from analytical and physicochemical data. The complexes are stable in solution as evidenced from the electrospray ionization mass spectrometry data. The complexes show good binding propensity with calf thymus (CT) DNA, giving binding constant (K-b) values of similar to 10(5) M-1 and a molecular ``light-switch'' effect that results in an enhancement of the emission intensity of the naphthalimide chromophore on binding to CT DNA. The complexes do not show any hydrolytic cleavage of DNA. They show poor chemical nuclease activity in the presence of 3-mercaptopropionic acid or hydrogen peroxide (H2O2). The Co(II) and Cu(II) complexes exhibit oxidative pUC19 DNA cleavage activity in UV-A light of 365 rim. The Zn(II) complex shows moderate DNA photocleavage activity at 365 nm. The Cu(II)complex 2 displays photoinduced DNA cleavage activity in red light of 647.1 nm and 676 rim and near-IR light of >750 rim. A mechanistic studyin UV-A and visible light suggests the involvement of the hydroxyl radical as the reactive species in the DNA photocleavage reactions. The complexes also show good bovine serum albumin (BSA) protein binding propensity, giving K-BSA values of similar to 10(5) M-1. Complexes 1 and 2 display significant photoinduced BSA cleavage activity in UV-A light. The Co(II) complex 1 shows a significant photocytotoxic effect in HeLa cervical cancer cells on exposure to UV-A light of 365 nm, giving an IC50 value of 32 mu M. The IC50 value for the py-nap ligand alone is 41.42 mu m in UV-A light. The IC50 value is >200 mu M in the dark, indicating poor dark toxicity of 1. The Cu(II) complex 2 exhibits moderate photocytotoxicity and significant dark toxicity, giving IC50 values of 18.6 mu m and 29.7 mu m in UV-A light and in the dark, respectively.

Solution NMR studies of acetohydroxy acid synthase I: Identification of the sites of inter-subunit interactions using multidimensional NMR methods

The novel multidomain organization in the multimeric Escherichia coli AHAS I (ilvBN) enzyme has been dissected to generate polypeptide fragments. These fragments when cloned, expressed and purified reassemble in the presence of cofactors to yield a catalytically competent enzyme. Structural characterization of AHAS has been impeded due to the fact that the holoenzyme is prone to dissociation leading to heterogeneity in samples. Our approach has enabled the structural characterization using high-resolution nuclear magnetic resonance methods. Near complete sequence specific NMR assignments for backbone H-N, N-15, C-13 alpha and C-13(beta) atoms of the FAD binding domain of ilvB have been obtained on samples isotopically enriched in H-2, C-13 and N-15. The secondary structure determined on the basis of observed C-13(alpha) secondary chemical shifts and sequential NOEs indicates that the secondary structure of the FAD binding domain of E. coli AHAS large Subunit (ilvB) is similar to the structure of this domain in the catalytic subunit of yeast AHAS. Protein-protein interactions involving the regulatory subunit (ilvN) and the domains of the catalytic subunit (ilvB) were studied using circular dichroic and isotope edited solution nuclear magnetic resonance spectroscopic methods. Observed changes in circular dichroic spectra indicate that the regulatory subunit (ilvN) interacts with ilvB alpha and ilvB beta domains of the catalytic subunit and not with the ilvB gamma domain. NMR chemical shift mapping methods show that ilvN binds close to the FAD binding site in ilvB beta and proximal to the intrasubunit ilvB alpha/ilvB beta domain interface. The implication of this interaction on the role of the regulatory subunit oil the activity of the holoenzyme is discussed. NMR studies of the regulatory domains show that these domains are structured in solution. Preliminary evidence for the interaction of ilvN with the metabolic end product of the pathway, viz., valine is also presented.

Encapsulation and release of rifampicin using poly(vinyl pyrrolidone)-poly (methacrylic acid) polyelectrolyte capsules

Poly(vinyl pyrrolidone) and poly(methacrylic acid) multilayer capsules based on hydrogen bonding have been prepared by the layer-by-layer approach and used to encapsulate and release rifampicin, an antituberculosis drug. Removal of silica core using a buffer of ammonium fluoride and hydrofluoric acid at about pH 3 was found to produce better capsules than hydrofluoric acid alone. An eight-layered capsule had a wall thickness of 20 rim. Maximum encapsulation was found to be about 86 mu g at 40 degrees C with 1 +/- 0.2 x 10(6) capsules. Release studies showed a burst kind of release and maximum release was obtained above pH 7 where the capsules disintegrate rapidly thereby releasing the drug in a short period. Interactions studies with Mycobacterium smegmatis showed that the capsules were cytocompatible and the released drug functioned with the same efficacy as the free drug.

Intensity Oscillation in the Corona as Observed during the Total Solar Eclipse of 29 March 2006

We obtained the images of the eastern part of the solar corona in the Fe xiv 530.3 nm (green) and Fe x 637.4 nm (red) coronal emission lines during the total solar eclipse of 29 March 2006 at Manavgat, Antalya, Turkey. The images were obtained using a 35 cm Meade telescope equipped with a Peltier-cooled 2k x 2k CCD and 0.3 nm pass-band interference filters at the rates of 2.95 s (exposure times of 100 ms) and 2.0 s (exposure times of 300 ms) in the Fe xiv and Fe x emission lines,respectively. The analysis of the data indicates intensity variations at some locations with period of strongest power around 27 s for the green line and 20 s for the red line. These results confirm earlier findings of variations in the continuum intensity with periods in the range of 5 to 56 s by Singh et al. (Solar Phys. 170, 235, 1997). The wavelet analysis has been used to identify significant intensity oscillations at all pixels within our field of view. Significant oscillations with high probability estimates were detected for some locations only. These locations seem to follow the boundary of an active region and in the neighborhood, rather than within the loops themselves. These intensity oscillations may be caused by fast magneto-sonic waves in the solar corona and partly account for heating of the plasma in the corona.