Purification and characterization of three toxins and two agglutinins from Abrus precatorius seed by using lactamyl-Sepharose affinity chromatography

Three toxins, abrin-I, -II, and -III, and two agglutinins, APA-I and -II, were purified from the seeds of Abrus precatorius by lactamyl-Sepharose affinity chromatography followed by gel filtration and DEAE-Sephacel column chromatography. abrin-I did not bind on DEAE-Sephacel column chromatography and the bound abrin-II, abrin-III, APA-I, and APA-II were eluted with a sodium acetate gradient. The identity of each protein was established by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The relative molecular weights are abrin-I, 64,000; abrin-II and abrin-III, 63,000 each: APA-I, 130,000; and APA-II, 128,000. Isoelectric focusing revealed microheterogeneity due to the presence of isoforms in each protein. Toxicity and binding studies further confirmed the differences among the lectins. The time course of inhibition of protein synthesis in thymocytes by the toxins showed lag times of 78, 61, and 72 min with Ki's of 0.55, 0.99, and 0.74 ms−1 at a 0.63 nImage concentration of each of abrin-I, -II, and -III, respectively. A Scatchard plot obtained from the equilibrium measurement for the lectins binding to lactamyl-Sepharose beads showed nonlinearity, indicating a cooperative mode of binding which was not observed for APA-I binding to Sepharose 4B beads. Further, by the criterion of the isoelectric focusing profile, it was shown that the least toxic abrin-I and the highly toxic abrin-II isolated by lactamyl-Sepharose chromatography were not retained on a low-affinity Sepharose 4B matrix, which signifies the necessity of using a high-affinity matrix for the purification of the lectins.

Plant aspartate transcarbamylase: An affinity chromatographic method for the purification of the enzyme from germinated seedlings

A simple and rapid affinity chromatographic method for the isolation of aspartate transcarbamylase from germinated seedlings of mung bean (Phaseolus aureus) was developed. A partially purified preparation of the enzyme was chromatographed on an affinity column containing aspartate linked to CNBr-activated Sepharose 4B. Aspartate transcarbamylase was specifically eluted from the column with 10 mImage aspartate or 0.5 Image KCl. The enzyme migrated as a single sharp band during disc electrophoresis at pH 8.6 on polyacrylamide gels. Electrophoresis of the sodium dodecyl sulfate-treated enzyme showed two distinct protein bands, suggesting that the mung bean aspartate transcarbamylase was made up of nonidentical subunits. Like the enzyme purified by conventional procedures, this enzyme preparation also exhibited positive homotropic interactions with carbamyl phosphate and negative heterotropic interactions with UMP. This method was extended to the purification of aspartate transcarbamylase from Lathyrus sativus, Eleucine coracona, and Trigonella foenum graecum.

Organometallic chemistry of diphosphazanes *1: VIII. Synthesis, spectroscopic studies and crystal structure of fac-[Mo(CO)3(MeCN)Ph2PN(iPr)P(Ph)(DMP)], and its reactions with tertiary phosphines

The reaction of fac-[Mo(CO)3(MeCN)3] with the unsymmetrical diphosphazane Ph2PN(iPr)P(Ph)(DMP) (L) gives the complex fac-[Mo(CO)3(MeCN)(L)] (2) in almost quantitative yield. The structure of the complex has been determined by an X-ray diffraction study. The compound reacts with PR3 (where R = Ph, OPh) to give fac-[Mo(CO)3(PR3)(L)] (3a, 4a), which undergoes an intramolecular isomerization to afford mer-[Mo(CO)3(PR3)(L)] (3b, 4b). Synthesis of cis-[Mo(CO)4(L)] (1) and fac-[MO(CO)3L] (2a) and their spectroscopic data are also reported.

Purification and Partial Characterization of Microsomal NADH-Cytochrome b5 Reductase from Higher Plant Catharanthus roseus

A simple three step procedure was used to purify microsomal NADH-cytochrome b5 (ferricyanide) reductase to homogeneity from the higher plant C. roseus. The microsomal bound reductase was solubilized using zwitterionic detergent-CHAPS. The solubilized reductase was subjected to affinity chromatography on octylamino Sepharose 4B, blue 2-Sepharose CL-6B and NAD+-Agarose. The homogeneous enzyme has an apparent molecular weight of 33,000 as estimated by SDS-PAGE. The purified enzyme catalyzes the reduction of purified cytochrome b5 from C. roseus in the presence of NADH. The reductase also readily transfers electrons from NADH to ferricyanide (Km 56 μM), 2,6-dichlorophenolindophenol (Km 65 μM) and cytochrome Image via cytochrome b5 but not to menadione.

Synthesis, Reactivity, Structural Aspects, and Solution Dynamics of Cyclopalladated Compounds of N,N `,N `'-Tris(2-anisyl)guanidine

N,N',N `'-Tris(2-anisyl)guanidine, (ArNH)(2)C=NAr (Ar = 2-(MeO)C6H4), was cyclopallaclated with Pd(OC(O)R)(2) (R = Me, CF3) in toluene at 70 degrees C to afford palladacycles Pd{kappa(2)(C,N)-C6H3-(OMe)-3(NHC(NHAr)(=NAr))-2}(mu-OC(O)R)](2)(R = Me (1a) and CF3 (1b)) in 87% and 95% yield, respectively. Palladacycle 1a was subjected to a metathetical reaction with LiBr in aqueous ethanol at 78 degrees C to afford palladacycle Pd{kappa(2)(C,N)-C6H3(OMe)-3(NHC(NHAr)(=NAr))-2}(mu-Br)](2) (2) in 90% yield. Palladacycle 2 was subjected to a bridge-splitting reaction with Lewis bases in CH2Cl2 to afford the monomeric palladacycles Pd{kappa(2)(C,N)-C6H3(OMe)-3(NHC(NHAr)(=NAr))-2}Br(L)] (L = 2,6-Me2C5H3N (3a), 2,4-Me2C5H3N (3b), 3,5-Me2C5H3N (3c), XyNC (Xy = 2,6-Me2C6H3; 4a), (BuNC)-Bu-t (4b), and PPh3 (5)) in 87-95% yield. Palladacycle 2 upon reaction with 2 equiv of XyNC in CH2Cl2 afforded an unanticipated palladacycle, Pd{kappa(2)(C,N)-C(=NXy)(C6H3(OMe)-4)-2(N=C-(NH Ar)(2))-3} Br(CNXy)] (6) in 93% yield, and the driving force for the formation of 6 was ascribed to a ring contraction followed by amine-imine tautomerization. Palladacycles 1 a,b revealed a dimeric transoid in-in conformation with ``open book'' framework in the solid state. In solution, 1 a exhibited a fluxional behavior ascribed to the six-membered ``(C,N)Pd'' ring inversion and partly dissociates to the pincer type and kappa(2)-O,O'-OAc monomeric palladacycles by an anchimerically assisted acetate cleavage process as studied by variable-temperature H-1 NMR data. Palladacycles 3a,b revealed a unique trans configuration around the palladium with lutidine being placed trans to the Pd-C bond, whereas cis stereochemistry was observed between the Pd-C bond and the Lewis base in 4a (as determined by X-ray diffraction data) and 5 (as determined by P-31 and C-13 NMR data). The aforementioned stereochemical difference was explained by invoking relative hardness/softness of the donor atoms around the palladium center. In solution, palladacycles 3a-c exist as a mixture of two interconverting boat conformers via a planar intermediate without any bond breaking due to the six-membered ``(C,N)Pd'' ring inversion, whereas palladacycles 4a,b and 5 exist as a single isomer, as deduced from detailed H-1 NMR studies.

Short-bite chiral diphosphazanes derived from (S)-?-methyl benzyl amine and their Pd, Pt and Rh metal complexes

New chiral diphosphazane ligands of the type Ph(2)PN(S-*CHMePh)PYY' {YY'= Ph(2) (2), O2C6H4 (3); Y= Ph, Y'= Cl {4a (SS), 4b (SR)}, N(2)C(3)HMe(2)-3,5 {5a (SR), 5b (SS)} are synthesised starting from a chiral aminophosphine, Ph(2)PNH(S-*CHMePh) (1). The structure of one of the diastereomer 5a has been confirmed by single crystal X-ray diffraction {Orthorhombic system, P2(1)2(1)2(1); a=10.456 (4), b=15.362 (7), c=17.379 (6) Angstrom, Z=4}. Transition metal mononuclear complexes [Rh{eta(2)-(Ph(2)P)(2)N- (S-*CHMePh)}(2)](+)(BF4)(-) (6), [PdCl2{eta(2)-(Ph(2)P)(2)N(S-*CHMePh)}] (7) and [PtCl2{eta(2)-(Ph(2)P)(2)N- (S-*CHMePh)}] (8) have also been synthesised. The structure of the palladium complex 7 is solved by X-ray crystallography {Orthorhombic system, P2(1)2(1)2(1); a=8.746 (2), b=18.086 (2), c=20.811 (3) Angstrom, Z=4}. All these compounds are characterised by micro analyses, IR and NMR spectroscopic data.

Purification and partial characterization of acyl carrier proteins from developing oil seeds of pisa (Actinodaphne hookeri) and ground nut (Arachis hypogaea)

Acyl carrier proteins (ACP) were purified to homogeneity in the active form from developing seeds of pisa (Actinodaphne hookeri) which synthesizes exclusively trilaurin and from ground nut (Arachis hypogaea) which synthesizes triacylglycerols containing long chain fatty acids. Two major isoforms of ACPs were purified from developing pisa seeds using DEAE-cellulose, Superose-6 FPLC and C-4 reversed phase HPLC chromatographic methods. In contrast, only a single form of ACP was present in ground nut seeds which was purified by anion-exchange and activated thiol-Sepharose 4B affinity chromatography. The two isoforms of ACPs from pisa showed nearly the same specific activity of 6,706 and 7,175 pmol per min per mg protein while ground nut ACP showed a specific activity of 3,893 pmol per min per mg protein when assayed using E. coli acyl-ACP synthetase and [1-C-14]palmitic acid. When compared with E. coli ACP, the purified ACPs from both the seeds showed considerable difference in their mobility in native PAGE, but showed similar mobility in SDS-PAGE under reducing conditions. In the absence of reducing agents formation of dimers was quite prominent. The ACPs from both the seed sources were acid- and heat-stable. The major isoform of pisa seed ACP and the ground nut ACP contain 91 amino acids with M(r) 11,616 and 1,228 respectively. However, there is significant variation in their amino acid composition. A comparision of the amino acid sequence in the N-terminal region of pisa and ground nut seed ACPs showed considerable homology between themselves and with other plant ACPs but not with E. coli ACP.

Synthesis, Structure, Negative Thermal Expansion, and Photocatalytic Property of Mo Doped ZrV(2)O(7)

A new series of compounds identified in the phase diagram of ZrO(2)-V(2)O(8)-MoO(3) have been synthesized via the solution combustion method. Single crystals of one of the compounds in the series, ZrV(1.50)Mo(0.50)O(7.25), were grown by the melt-cool technique from the starting materials with double the MoO(3) quantity. The room temperature average crystal structure of the grown crystals was solved using the single crystal X-ray diffraction technique. The crystals belong to the cubic crystal system, space group Pa (3) over bar (No. 205) with a = 8.8969 (4) angstrom, V = 704.24 (6) angstrom(3), and Z = 4. The final R(1) value of 0.0213 was achieved for 288 independent reflections during the structure refinement. The Zr(4+) occupies the special position (4a) whereas V(5+) and Mo(6+) occupy two unique (8c) Wyckoff positions. Two fully occupied O atoms, (24d) and (4b), one partially occupied 0 atom (8c) have been identified for this molybdovanadate, which is a unique feature for these crystals. The structure is related to both ZrV(2)O(7) and cubic ZrMo(2)O(8). The temperature dependent single crystal studies show negative thermal expansion above 370 K. The compounds have been characterized by powder X-ray diffraction, solid-state UV-vis diffuse reflectance spectra, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The photocatalytic activity of these compounds has been investigated for the degradation of various dyes, and these compounds show specificity toward the degradation of non-azoic dyes.

Hybrid molecules of carvacrol and benzoyl urea/thiourea with potential applications in agriculture and medicine

Benzoyl phenyl urea, a class of insect growth regulator's acts by inhibiting chitin synthesis. Carvacrol, a naturally occurring monoterpenoid is an effective antifungal agent. We have structurally modified carvacrol (2-methyl-5-1-methylethyl] phenol) by introducing benzoylphenyl urea linkage. Two series of benzoylcarvacryl thiourea (BCTU, 4a-f) and benzoylcarvacryl urea (BCU, 5a-f) derivatives were prepared and characterized by elemental analysis, IR, H-1 and C-13 NMR and Mass spectroscopy. Derivatives 4b, 4d, 4e, 4f and 5d, 5f showed comparable insecticidal activity with the standard BPU lufenuron against Dysdercus koenigii. BCTU derivatives 4c, 4e and BCU 5c showed good antifungal activity against phytopathogenic fungi viz. Magnaporthe grisae, Fusarium oxysporum, Dreschlera oryzae; food spoilage yeasts viz. Debaromyces hansenii, Pichia membranifaciens; and human pathogens viz. Candida albicans and Cryptococcus neoformans. Compounds 5d, 5e and 5f showed potent activity against human pathogens. Moderate and selective activity was observed for other compounds. All the synthesized compounds were non-haemolytic. These compounds have potential application in agriculture and medicine. (C) 2012 Elsevier Ltd. All rights reserved.

Analysis of designed beta-hairpin peptides: molecular conformation and packing in crystals

The crystal structures of several designed peptide hairpins have been determined in order to establish features of molecular conformations and modes of aggregation in the crystals. Hairpin formation has been induced using a centrally positioned (D)Pro-Xxx segment (Xxx = (L)Pro, Aib, Ac(6)c, Ala; Aib = alpha-aminoisobutyric acid; Ac(6)c = 1-aminocyclohexane-1-carboxylic acid). Structures of the peptides Boc-Leu-Phe-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe (1), Boc-Leu-Tyr-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe (2, polymorphic forms labeled as 2a and 2b), Boc-Leu-Val-Val-(D)Pro-(L)Pro-Leu-Val-Val-OMe (3), Boc-Leu-Phe-Val-(D)Pro-Aib-Leu-Phe-Val-OMe (4, polymorphic forms labeled as 4a and 4b), Boc-Leu-Phe-Val-(D)Pro-Ac(6)c-Leu-Phe-Val-OMe (5) and Boc-Leu-Phe-Val-(D)Pro-Ala-Leu-Phe-Val-OMe (6) are described. All the octapeptides adopt type II' beta-turn nucleated hairpins, stabilized by three or four cross-strand intramolecular hydrogen bonds. The angle of twist between the two antiparallel strands lies in the range of -9.8 degrees to -26.7 degrees. A detailed analysis of packing motifs in peptide hairpin crystals is presented, revealing three broad modes of association: parallel packing, antiparallel packing and orthogonal packing. An attempt to correlate aggregation modes in solution with observed packing motifs in crystals has been made by indexing of crystal faces in the case of three of the peptide hairpins. The observed modes of hairpin aggregation may be of relevance in modeling multiple modes of association, which may provide insights into the structure of insoluble polypeptide aggregates.

Insertion Reactions of Six-Membered Cyclopalladated N,N `,N `'-Triarylguanidine, Pd{kappa(2)(C,N)-C6H3Me-3(NHC(NHAr)(=NAr))-2)(mu-Br)](2) (Ar=2-MeC6H4) with PhCC C-C(O)OR (R = Me and Et): A Gateway to Second Orthopalladation through Novel Rearrangements

The reaction of Pd{kappa(2)(C,N)-C6H3Me-3-(NHC(NHAr)(=NAr))-2}(mu-Br)](2) (Ar = 2-MeC6H4; 1) with 4 equiv of PhC C-C(O)OMe in CH2Cl2 afforded Pd{kappa(2)(C,N)-C(Ph)=C(C(O)OMe)C(Ph)=C(C(O)-OMe)C6H3Me-3(N=C(NH Ar)(2))-2}Br] (Ar = 2-MeC6H4; 2) in 70% yield, and the aforementioned reaction carried out with 10 equiv of PhC C-C(O)OR (R = Me, and Et) afforded an admixture of two regioisomers of Pd{kappa(3)(N,C,O)-O=C(OR)-C5Ph3(C(O)OR)C(C(O)OR)C6H3Me-3(N=C(NHAr)( 2))- 2}Br] (Ar = 2-MeC6H4; R = Me (3a/3b), Et (4a/4b)) in 80 and 87% yields, respectively. In one attempt, the minor regioisomer, 4b, was isolated from the mixture in 6% yield by fractional crystallization. Palladacycles 3a/3b and 4a/4b, upon stirring in CH2Cl2/MeCN (1/1, v/v) mixture at ambient condition for S days, afforded Pd{eta(3)-allyl,(KN)-N-1)-C-5(C(O)OR)(2)Ph3C-(C(O)OR)C6H3Me-3(N=C(NH Ar)(2))(-2)}Br] (Ar = 2-MeC6H4; R = Me (5a/5b), Et (6a/6b)) in 94 and 93% yields, respectively. Palladacycles 3a/3b and 4a/4b, upon reaction with AgOTf in CH2CH2/Me2C(O) (1/1, v/v) mixture at ambient temperature for 15 min, afforded Pd{kappa(3)(N,C,O)-O=C(OR)C5Ph3(C(O)OR)C(C(O)OR)C6H3Me-3(N=C(NHAr)(2 ))-2}(OTf)] (Ar = 2-MeC6H4; R = Me (7a/7b), Et (8a/8b)) in 79 and 77% yields, respectively. Palladacycles 7a/7b and 8a/ 8b, upon reflux in PhC1 separately for 6 h, or palladacycles 5a/5b and 6a/6b, upon treatment with AgOTf in CH2Cl2/Me2C(O) (7/3, v/v) mixture for 15 min, afforded Pd{(eta(2)-Ph)C5Ph2(C(O)OR)kappa(2)(C,N)-C(C(O)OR)C6H3Me-3(N=C(NHAr) (2))-2}(OTf)] (Ar = 2-MeC6H4; R = Me (9a/9h), Et (10a/10b)) in >= 87% yields. Palladacycles 9a/9b, upon stirring in MeCN in the presence of excess NaOAc followed by crystallization of the reaction mixture in the same solvent, afforded Pd{kappa(3)(N,C,C)-(C6H4)C5Ph2(C(O)OMe)(2)C(C(O)OMe)(2)C6H3Me-3(N=C( NHAr)(2))-2}(NCMe)] (Ar = 2-MeC6H4; 11a/11b) in 82% yield. The new palladacycles were characterized by analytical, IR, and NMR (H-1 and C-13) spectroscopic techniques, and the molecular structures of 2, 3a, 4a, 4b, 5a, 6a, 7a, 9a, 10a, and 11a-d(3) were determined by single crystal X-ray diffraction. The frameworks in the aforementioned palladacycles, except that present in 2, are unprecedented. Plausible pathways for the formation of new palladacycles and the influence of the guanidine unit in 1, substituents in alkynes, reaction conditions, and electrophilicity of the bromide and the triflate upon the frameworks of the insertion products have been discussed.

Novel 2,6-bis(4-methoxyphenyl)-1-methylpiperidin-4-one oxime esters: synthesis and a new insight into their antioxidant and antimicrobial potential

A simple and efficient protocol for the synthesis of novel 2,6-bis(4-methoxyphenyl)-1-methylpiperidin-4-one oxime esters 4(a-q) is described. Initially, p-anisaldehyde 1 was condensed (Mannich reaction) with acetone and ammonium acetate trihydrate afforded 2,6-bis(4-methoxyphenyl)piperidin-4-one 2. Then, methylation followed by oximation with hydroxylamine hydrochloride (NH(2)OHa (TM) HCl) furnished a key scaffold 4. Further, to explore the enhanced biological properties of the piperidin-4-one core i.e. the key scaffold 4 was conjugated with substituted benzoyl chlorides in the presence of anhydrous K2CO3 as base to obtain novel 2,6-bis(4-methoxyphenyl)-1-methylpiperidin-4-one oxime esters 4(a-q) in excellent yields. The newly synthesized compounds were characterized by elemental analysis, IR, H-1 NMR, C-13 NMR and mass spectroscopic techniques, and screened for their in vitro antioxidant and antimicrobial activities. Most of the compounds exerted positive efficacy towards the biological assays performed. Among the synthesized analogues, compounds 4l and 4m exhibited promising antioxidant activity and on the other hand compounds 4b and 4d manifested persuasive antibacterial activity, whereas compound 4b displayed stupendous antifungal activity against A. flavus strain.

New columnar liquid crystal materials based on luminescent 2-methoxy-3-cyanopyridines

A new series of donor-acceptor-donor (D-A-D) type luminescent mesogens carrying 2-methoxy-3-cyanopyridine as a central core linked with variable alkoxy chain lengths (m = 6 and 8) as terminal substituents was synthesized and characterized using spectral methods. The newly synthesized molecules were subjected to single-crystal X-ray diffraction (SCXRD), powder X-ray diffraction (PXRD), differential scanning calorimetric (DSC), polarizing optical microscopy (POM), and fluorescence emission studies in order to ascertain their mesogenic and photophysical properties. The SCXRD data on 4a and 4b reveal that the presence of short intermolecular contacts, viz. C-H center dot center dot center dot N, C-H center dot center dot center dot O, C-H center dot center dot center dot pi, and pi center dot center dot center dot pi interactions, is responsible for their crystal packing. The measured torsion angle values indicate that molecules possess distorted non-planar structure. The DSC, POM, and PXRD studies confirm that all the molecules show thermotropic liquid crystalline behaviour and exhibit rectangular columnar phase. Further, their UV-visible and fluorescence spectral studies reveal that the target molecules are luminescent displaying a strong absorption band in the range of 335-340 nm and a blue fluorescence emission band in the range of 395-425 nm (both in solution and film state) with good fluorescence quantum yields (10-49 %).

A Multiantigenic DNA Vaccine That Induces Broad Hepatitis C Virus-Specific T-Cell Responses in Mice

There are 3 to 4 million new hepatitis C virus (HCV) infections annually around the world, but no vaccine is available. Robust T-cell mediated responses are necessary for effective clearance of the virus, and DNA vaccines result in a cell-mediated bias. Adjuvants are often required for effective vaccination, but during natural lytic viral infections damage-associated molecular patterns (DAMPs) are released, which act as natural adjuvants. Hence, a vaccine that induces cell necrosis and releases DAMPs will result in cell-mediated immunity (CMI), similar to that resulting from natural lytic viral infection. We have generated a DNA vaccine with the ability to elicit strong CMI against the HCV nonstructural (NS) proteins (3, 4A, 4B, and 5B) by encoding a cytolytic protein, perforin (PRF), and the antigens on a single plasmid. We examined the efficacy of the vaccines in C57BL/6 mice, as determined by gamma interferon enzyme-linked immunosorbent spot assay, cell proliferation studies, and intracellular cytokine production. Initially, we showed that encoding the NS4A protein in a vaccine which encoded only NS3 reduced the immunogenicity of NS3, whereas including PRF increased NS3 immunogenicity. In contrast, the inclusion of NS4A increased the immunogenicity of the NS3, NS4B, andNS5B proteins, when encoded in a DNA vaccine that also encoded PRF. Finally, vaccines that also encoded PRF elicited similar levels of CMI against each protein after vaccination with DNA encoding NS3, NS4A, NS4B, and NS5B compared to mice vaccinated with DNA encoding only NS3 or NS4B/5B. Thus, we have developed a promising ``multiantigen'' vaccine that elicits robust CMI. IMPORTANCE Since their development, vaccines have reduced the global burden of disease. One strategy for vaccine development is to use commercially viable DNA technology, which has the potential to generate robust immune responses. Hepatitis C virus causes chronic liver infection and is a leading cause of liver cancer. To date, no vaccine is currently available, and treatment is costly and often results in side effects, limiting the number of patients who are treated. Despite recent advances in treatment, prevention remains the key to efficient control and elimination of this virus. Here, we describe a novel DNA vaccine against hepatitis C virus that is capable of inducing robust cell-mediated immune responses in mice and is a promising vaccine candidate for humans.