Bovine herpesvirus 1 DNA has been isolated by SDS lysis of the virus purified from potassium tartrate (10-50%) density gradient centrifugation. The quality and quantity of viral DNA was checked by UV spectrophotometry and ethidium bromide stained agarose gel electrophoresis. The 0.4 kb Hin dIII'O' fragment of BHV-1 DNA was selectively cloned into Hin dIII cut pUC9 plasmid DNA (2.665 kb). Recombinants were screened by white/blue colonies as well as Hin dIII restriction enzyme analysis. On restriction endonuclease analysis of recombinant plasmid DNA (p-BH-0) with several restriction enzymes, viz., Sau 3A, Hin fI, Rsa I, Sal I, Dra I, Bgl I, Bgl II, Sma I, Hpa I, Stu I, Mlu I, Xho I, Kpn I, Hae III, Eco RI, Bam HI, Pst I, Pal I, revealed insert viral DNA having sites for Hin fI, Hae III, Rsa I, Sma I, only. Further, the partial restriction map of the recombinant plasmid DNA was constructed using above enzymes.
Considering the potential of peripheral benzodiazepine receptor (PBR) ligands in therapeutic applications and clinical benefit in the management of a large spectrum of different indications, quantitative structure-activity relationship (QSAR) study has been attempted to explore the structural and physicochemical requirements for selectivity of 2-phenylimidazo[1,2-a]pyridineacetamides for binding with peripheral over central benzodiazepine receptors (CBRs). For PBR binding affinity, molar refractivity (MR) shows a parabolic relation with binding affinity suggesting that binding affinity increases with increase in volume of the compounds, until it reaches the critical value, after which the affinity decreases. The negative coefficients of S_aaN and S_ssNH indicate that binding affinity increases with decrease in E-state value of (N/) (aromatic nitrogen) and HN< (secondary amino group) fragments. The coefficient of 3XVC and JX term indicates the importance of shape and branching for binding affinity. For CBR binding affinity, lipophilicity of molecules is detrimental to the binding affinity, while presence of hydrogen at Y position is conducive to the activity. Selectivity pattern of these ligands for peripheral (cortex) over central receptors requires the presence and absence of methyl group at R2 and R3 positions respectively, and shows the importance of MR and shape parameter. Similarly, selectivity of these ligands for peripheral (ovary) over central receptors requires the presence and absence of methyl group at R2 and R3 positions respectively, presence of phenyl group at R1 and R2 positions and selectivity relation shows importance of MR, shape and branching.
Molecular dynamics (MD) simulation of the interaction between amphiphilic polypeptide Ac(LKKL)4NHEt and 4 DMPC (1,2 di-mysristoyl-sn-glycero-3-phosphorylcholine) molecules has been carried out at 310 K for 500 picoseconds (ps) using AMBER 4.0. Interaction energy and a number of conformational parameters are calculated for the subaveraged coordinates, using P-CURVES 3.1 and our MD trajectory analysis program ANALMD. No significant change in DMPC headgroup conformation was observed. However, the mobility of P atoms was found to be restricted. The chains were quite flexible and their flexibility increased towards the ends. They interacted amongst themselves. The polypeptide remained predominantly in alpha-helical conformation. Leu1 and Lys2 at the N terminus and Leu13 to Leu16 at C terminus assumed non helical conformation and were quite flexible. Average interaction energy between the polypeptide and DMPC molecules was found to be -151.828 kcal*mol-1. The main contributory factor was electrostatic interaction of Lys NH3+ groups with the DMPC phosphates. On an average one Lys chain interacted with 1.5 DMPC molecules. Central region of the polypeptide had better contact with DMPC molecules. A model for the fusogenic properties of the polypeptide is presented on the basis of MD results.
Considering importance of developing selective COX-2 inhibitors, COX-2 binding affinity data of 4-(2-aryl-1-imidazolyl)-phenyl methyl sulfones and sulfonamides (n = 83) have been modeled using electrotopological state (E-state) index as electronic parameter, hydrophobic substituent constant (pi) and molar refractivity (MR) of aryl ring substituents as lipophilic and steric parameters, respectively. Additionally, suitable dummy parameters have been used for the development of multiple regression equations in a stepwise manner. The study suggests that lipophilicity of ortho, meta and para substituents of the aryl ring increases the binding affinity, while molar refractivity (MR) of ortho and meta substituents of the aryl ring decreases the binding affinity. Again, electron-withdrawing substituents at meta and para positions of the aryl ring increase the binding affinity. Additionally, a 4-fluoro substituent on the aryl ring, a trifluoromethyl substituent at R position and simultaneous presence of 3-chloro and 4-methyl groups on the aryl ring are conducive to the binding affinity. Also, an amino substituent is preferred over a methyl group at R2 position suggesting preference of the sulfonamide moiety over the methyl sulfone moiety for the COX-2 binding affinity. Furthermore, importance of E-state values of different atoms in the generated relations suggests the influence of electron density distribution over the 1,2-diarylimidazole nucleus for the binding affinity. For this data set, E-state parameters perform better as electronic parameters in comparison to Hammett sigma parameters. When lipophilic whole molecular descriptor (ClogP) is used, instead of hydrophobic substituent constant (pi), the former performs better than the latter.
Carbonic anhydrase (CA) inhibitors are very interesting target for designing anticancer (hypoxic) and antiglaucoma drugs. In the present study, a 3D homology modeling of human carbonic anhydrase-IX (hCA-IX) isozyme, based upon the crystal structure of murine CA-XIVA (PDB CODE 1RJ5) was performed, as no experimental 3D structures are available. A homology model of hCA-IX was developed and validated. To explore the responsible physicochemical properties of 1,3,4-thiadiazole and 1,3,4-triazole derivatives for carbonic anhydrase inhibition, a quantitative structure activity relationship (QSAR) study was performed having hCA-II and hCA-IX inhibitory activity respectively. In hCA-II and hCA-IX inhibitory activities, four significant models with good correlations (> or = 0.945 & > or = 0.926) were obtained; two models (models 1 and 3) were selected based on statistical criterion. The QSAR study revealed that in case of hCA-II, overall increase in size and volume of molecule, introduction of electropositive surfaces might increase the inhibitory activity, whereas in case of hCA-IX, decreasing the hydrophobicity and introduction of electron releasing substituents might increase the hCA-IX inhibitory activity.
Electron transport activity of beet spinach thylakoids was enhanced in the presence of aliphatic amine, DABCO (1,4-diazobicyclo (2,2,2) octane), a hydrophilic proton trapping agent. The extent of stimulation was pH-dependent and similar to the effect of the uncoupler ammonium chloride on electron transport. The stimulation of whole-chain (H2O-->MV) electron transport activity was observed only at high (rate-saturating) light intensity. The light-induced proton uptake coupled to electron transport of thylakoid was also arrested by DABCO, suggesting that DABCO uncouples thylakoid phosphorylation by proton trapping.
Antimicrobials derived from plants have been receiving increasing attention in recent years. Antimicrobial activities of a number of phytochemicals have been reported. Many present day antibiotics are ineffective against several pathogenic organisms. About 90% of Staphylococcus aureus isolates from clinical specimens is reported to have resistance against beta-lactam antibiotics. In the present study, the effect of hexane, diethyl ether, acetone and water extracts of leaves of a medicinal plant Holoptelea integrifolia has been tested against beta-lactam resistant strain of S. aureus in presence of antibiotics such as ampicillin, amoxicillin, cefotaxime and ceftriaxone. The diethyl ether extract has shown the maximum antibacterial activity and the active principle is found to be 1,4-naphthalenedione which is characterized by GC-MS and FTIR spectroscopy. The minimum inhibitory concentration (MIC) of the compound is found to be 4 mg/ml. Structural similarity of this compound with a functional group of a beta-lactamase-resistant antibiotic indicates that 1,4-naphthlenedione may be acting as an inhibitor to beta-lactamase.
The energetics of the precipitation process depended on the solute-solvent combination and the enthalpy and entropy of precipitation compensated each other. The partial molal volumes of the lipids in both the solvents were greater than the anhydrous molar volumes, except for cholesterol in 1,4-dioxane and cholesteryl acetate in 2-propanol where the order was reverse. While the partial molal compressibilities of all the solutes studied were negative in 1,4-dioxane, those of cholesterol and cholesteryl acetate in 2-propanol were, respectively, negative and positive. The negative values were supported by considerable solvation of the solutes, particularly in 1,4-dioxane.
Phytase is a monomeric enzyme of molecular mass 160 kDa which catalyzes the hydrolysis of phytic acid (D-myo inositol hexakisphosphate, InsP6) in a stepwise manner to myo-inositol. The enzyme-InsPn (n = 1-6) interaction at the catalytic site has a dissociation constant in the micro molar range. There also exists in the enzyme, a non-catalytic site specific for InsP3 with dissociation constant in the nano molar range. We have probed the effect of the high affinity InsP3 binding on the dissociation constant (Kd) of the phytase-InsP6 interaction and the kinetics of hydrolysis. These studies demonstrate the effect exerted by the high affinity InsP3 binding on the catalytic site of the enzyme.
Ribulose-1,5-bisphosphate carboxylase has been isolated from a synthetic cereal triticale and purified using a newly developed rapid procedure involving precipitation with ammonium sulphate (35-55% saturation), DEAE-cellulose (DE-52) chromatography and filtration through Sepharose CL-68. Molecular weights of the enzyme subunits are 15.5 and 52 kDa which corresponds to 540 kDa for the hexadecameric holoenzyme. Isoelectric focussing showed that the enzyme has a pI of 4.2. Various kinetic constants determined under aerobic conditions are: Km (CO2), 118 microM; Km (RuBP), 220 microM (at 20 mM NaHCO3) and Vmax, 690 nmole CO2 fixed/mg enzyme/min.
Ribulose 1,5-bisphosphate carboxylase [3-phospho-D-glyceratecarboxy-lyase (dimerizing), EC 220.127.116.11] is rapidly and irreversibly inactivated by micromolar concentrations of dimethyl (2-hydroxy-5-nitrobenzyl) sulphonium bromide (DMHNB), a tryptophan selective reagent, after reversible protection of the reactive sulphydryl groups. The inactivation followed pseudo-first-order reaction kinetics. Replots of the kinetic data indicated that no reversible enzyme-inhibitor complex was formed prior to irreversible modification. Kinetic analysis and the correlation of the spectral data at 410 nm with enzyme activity indicated that inactivation by DMHNB resulted from modification of on an average one tryptophan per 67 kDa combination of large and small subunits. Several competitive inhibitors and substrate RuBP offered strong protection against inhibition. The k1/2 (protection) for RuBP was 1.3 mM, indicating that the tryptophan residues may be located at or near the substrate binding site. Free and total sulphydryl groups were not affected by the reagent. The modified enzyme exhibited significantly reduced intrinsic fluorescence, indicating that the microenvironment of the tryptophans at the active site is significantly perturbed. Tryptic peptide profiles and CD spectral analyses suggested that inactivation may not be due to the extensive conformational changes in the enzyme molecule during modification.
Fructose diphosphate aldolase has been purified 64 fold from surface grown Mycobacterium tuberculosis H37Rv. The aldolase is a typical class II type and is inhibited by EDTA and o phenanthroline. K+ and Mg2+ stimulated the enzyme activity and pyrophosphate was a potent inhibitor. Cysteine, glutathione and dithiothreitol enhanced the activity of the enzyme, suggesting a requirement for -SH group.
Teichoic acid (TA) isolated from the Gram-positive bacteria Staphylococcus aureus induces blue shifted metachromasia in the dye 1,9-dimethyl methyleneblue (DMMB). Results of conductometric titrations show that DMMB binds to TA with 1:1 stoichiometry. Unlike DNA, this TA does not induce a definite metachromatic band in the dye acridine orange. S. aureus TA also induces strong circular dichroism (CD) in this dye with molar ellipticity value of the order of 10(4). This induction of CD in DMMB supports our earlier inference that TA in dilute solution at neutral pH has a helical conformation, and is not a random coil polymer.
The theoretical prediction of induction of metachromasia [V Czikkely, H D Foersterling & H Kuhn (1970), Chem Phys Lett, 6,207] in a dye by a polyanion having only four to six anionic sites is proved experimentally, for the first time, in ATP--1.9-dimethyl methylene blue system. The findings show that ATP induces metachromasia in the dye at neutral pH, when ATP molecule remains fully charged providing four anionic sites to the dye cations. Conductometric titration shows that the dye molecules bind stoichiometrically to ATP (four dyes/ATP). However ATP at acidic pH and ADP and AMP at any pH fail to induce metachromasia. This is also the first report of induction of circular dichroism in bound dyes by ATP. Though the chiral moiety of ribose sugar in ATP may induce dichroism in the bound achiral dyes, the observed high molar ellipticity values indicate aggregation of bound dyes with twist in one sense initiated by the twisted conformation of the triphosphate chain in ATP. This inference on the state of conformation of ATP in its native environment is in agreement with that derived from PMR and spin lattice relaxation technique. It is thus interesting that the conformation of crystalline disodium ATP, as concluded from X-ray crystallography, is maintained by tetrasodium ATP in dilute aqueous solution--the native environment of ATP.
Secretory phospholipase A2s (PLA2s), the structurally-homologous enzymes share a common qualitative catalytic site, but differ greatly in their pharmacological properties and toxicities. There has been a recognizable pattern of mutations in the primary sequence of PLA2s that alter their catalytic properties significantly. In the present study, the amino acid sequence and the three-dimensional structure of a new isoform of PLA2 from crude venom of Indian cobra sub-species Naja naja sagittifera (N.n.s.) has been determined by X-ray crystallography. The crystal structure has revealed several novel features of PLA2 folding and function. It contains 913 protein atoms and one each of Ca2+, phosphate and acetate ions with 142 solvent water molecules. A Ca2+ ion is present in the calcium-binding loop and forms a seven-fold coordination with a distorted pentagonal bipyramidal geometry. One of the coordination linkages is with the acetate ion, instead of the conserved water molecule. The presence of Lys at position 31 has a stabilizing effect on the loop Tyr 25-Cys 29 by interacting with carbonyl oxygen atoms of Tyr 25, Gly 26 and Cys 29. In turn, it lends stability to the Ca(2+)-binding loop as well. Another unique feature of the PLA2 structure is the formation of an intrastrand hydrogen bond, involving Ogamma of Thr 73 and Oepsilon2 of Glu 71, thus helping the beta-wing to act as a sharp arrow for insertion into other molecules. Yet another important feature of this PLA2 pertains to the conformation of its C-terminal segment, which is stabilized by a unique hydrogen bond through carbonyl oxygen of Lys 116 and Ndelta2 of Asn 120. This structural feature may be useful in the molecular recognition of the PLA2 through C-terminal segment.
Aeromonas caviae W-61 produces multiple extracellular xylanases, the xylanases 1, 2, 3, 4, and 5. In this study, we purified and characterized the xylanase 5 of A. caviae W-61, and amplified a part of xylanase 5 gene (xyn5). The purified xylanase 5 was found to be a single polypeptide with molecular mass of 140 kDa. It was an endo-beta-1,4-xylanase showing optimum temperature 40 degrees C and optimum pH 6.0. Xylobiose, xylotriose, xylotetrose, xylopentose, xylohexose and a small amount of xylose were detected as the hydrolysis products. The N-terminal amino acid sequence and several internal amino acid sequences of xylanases 5 were determined. From the sequence, a 1.8 kbp fragment was amplified by PCR using forward and reverse primers. DNA sequencing confirmed the presence of nucleotide sequences corresponding to the N-terminal amino acid sequence and the internal amino acid sequences of xylanase 5.
Proteinese K (PK) isolated from Tritirachium album Limber was crystallized with HgCl2 in excess, under microgravity conditions. The intensity data were collected at 4 degrees C to 1.8 A resolution and the final R-factor after refinement for all the reflections was 0.164. Mercury has been found at two sites with partial occupancies (0.4 and 0.6) which are at distances of 2.48 A and 2.58 A respectively from Cys-73 Sgamma. The Cys-73 in the enzyme structure is located close to the active site residue, His-69. This region is completely buried and is not accessible to the solvent. It is rather tightly packed. Therefore, the binding of mercury distorts the stereochemistry of the neighbouring residues including those belonging to the catalytic triad. As a result of this, the Ogamma of Ser-224 is displaced by 0.6 A which causes the inactivation of proteinase K by increasing the H-bond distance to 3.7 A between Ser-224 Ogamma and His-69 Nepsilon2.
An oxalate oxidase was purified to apparent homogeneity from the leaves of 10-days old seedlings of forage Sorghum (Sorghum vulgare var. KH-105). The enzyme had a Mr of 124 kDa with two identical subunits, an optimum pH of 4.5, optimum temperature of 37 degrees C and activation energy (Ea) of 2.0338 Kcal/mol. The rate of reaction was linear up to 7 min. K(m) value for oxalate was 0.22 mM. The enzyme was stimulated by Cu2+ and inhibited by EDTA, NaCN, diethyldithiocarbamate, Na2SO4, but unaffected by NaCl at 0.1 mM concentration. Although the enzyme was stimulated by flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), UV and visible spectra of the enzyme did not match with that of a flavoprotein. The positive reaction of the enzyme with orcinol-H2SO4 reagent indicated its glycoprotein nature. The superiority of the purified enzyme over earlier reported oxalate oxidases for determination of urinary oxalate has been demonstrated.
Candida 107 (NCYC 911) accumulates up to 45% of the biomass as triglycerides under conditions of nitrogenous substrate limitation in the medium. In oilseeds and adipocytes, lipid accumulation is preceded and accompanied by increased activity of key enzymes such as pyruvate dehydrogenase. However, in Candida 107, the activity of this complex was greatly reduced during lipogenesis. The initial velocity patterns were in accordance with a Hexa Uni Ping Pong mechanism. The Km values for the various substrates were similar to those found for the yeast Saccharomyces cerevisiae, but much higher than those reported for the mammalian enzyme. Product inhibition studies indicated that the Ki for acetyl coenzyme A and NADH were higher than those reported for other yeasts. The values for Ki were similar to those found for the liver enzyme, whereas the enzyme complex from heart had much lower Ki values for products. It has been suggested that in the heart and kidney, pyruvate dehydrogenase is regulated by product inhibition whereas in the liver this does not appear to be the mechanism. Therefore, it is probable, that like the liver enzyme, pyruvate dehydrogenase from Candida 107 may not be regulated by product inhibition.
Substance P belongs to the tachykinin family of neuropeptides which exhibit diverse pharmacological activity. The conformation of Phe1-Phe2-Gly3-Leu4-Met5-NH2 the C-terminal pentapeptide of substance P (SP7-11) has been studied by NMR and molecular dynamics (MD) methods. NMR studies were carried out both in DMSO-d6 and 95% H2O. Based on the observed chemical shifts, 3JNH alpha coupling constants, temperature coefficients of chemical shifts of NH resonances and the pattern of inter- and intraresidue NOE's, a predominantly extended backbone conformation has been deduced for the peptide in both DMSO and H2O. MD calculations carried out in vacuo indicate that the global minimum energy conformation of the molecule is folded with an intramolecular hydrogen bond between the protonated N-terminal and the C-terminal CONH2 group. The simulation shows that beta-turns are energetically unfavourable, while alpha-helices are seen to be unstable for the peptide. gamma-Bends at either Gly3 or Leu4 are the most preferred ones. Simulations carried out in DMSO as well as in water show a preference for a nearly extended conformation.
An antileishmanial compound, 14-deoxy-11-oxo-andrographolide, a derivative of andrographlide, isolated from the Indian medicinal plant Andrographis paniculata was evaluated for efficacy in free form and in different vesicular delivery modes on hamster model of Leishmaniasis. The subcutaneous injection of free drug reduced the spleen parasite load by 39%, whereas for drug incorporated in liposomes, niosomes and microspheres, reductions in the parasite load were 78%, 91% and 59%, respectively. Moreover, the drug in various delivery modes, particularly in liposomal and niosomal forms, showed no apparent immediate toxicity. Although an inverse linear relationship between the size of carriers and per cent efficacy in reduction of spleen parasite load was established, involvement of other factors such as drug release profiles or rates remains an open question. Because of greater efficacy and lesser toxicity, liposomal, niosomal and possibly microsphere-incorporated 14-deoxy-11-oxo-andrographolide might have clinical application to combat visceral Leishmaniasis.
A method has been developed for preparing 125I-insulin of long shelf life and high immunogenicity. The technique is based on the use of chloramine-T as an oxidizing agent and Sephadex column chromatography for separating unreacted 125I from 125I-insulin. The fraction shown the maximum amount of labelled hormone, as indicated from measurement of radioactivity in each of the fractions eluted from the column by barbitone buffer (0.1 M, pH 8.6), was further purified by passing through cellulose column and eluting with 0.15 N HCl-EtOH (25:75, vol./vol.) solvent mixture. Adsorption on dextran-coated charcoal, precipitation by 10% trichloracetic acid and binding with excess of antibodies to insulin were carried out with and without repurification of 125I-insulin by cellulose column chromatography. The purified preparation of radioiodinated insulin was found highly promising for the purpose of radioimmunoassay of insulin in human blood samples and plasma membrane receptor binding studies for a period of at least 7 weeks whereas 125I-insulin without such repurification lost its usefulness within a period of 2 weeks.
In view of documented evidence demonstrating the association of dopaminergic metabolism and neurotransmission with Parkinson's disease (PD), a case-control study was conducted to investigate the impact of particular polymorphisms in the catechol O-methyl transferase (COMT) H108L, monoamine oxidase B (MAOB) int 13 A>G, dopamine transporter 1 (DAT1) A1215G, dopamine receptor D2 (DRD2) Taq1A, DRD2 Taq1B and DRD2 Taq1D genes on the susceptibility to PD. PCR-RFLP method was used for the genetic analysis. The COMT H108L polymorphism increased PD risk by 1.4-fold (95%CI: 1.02-1.98), whereas reduced risk was observed with MAOB int 13 A>G polymorphism (OR: 0.77, 95%CI: 0.51-0.99). Multifactor dimensionality reduction analysis showed gene-gene interactions between these two loci that resulted in loss of the protective role of MAOB G-allele in the presence of COMT L-allele. DAT1A1215G polymorphism in the exon 9 was not associated with PD. Individually, DRD2 polymorphisms showed null association. However, all-variant haplotype of DRD2 locus i.e. T-G-T haplotype showed 29.8-fold risk for PD compared to all-wild haplotype i.e., C-A-C haplotype (95%CI: 6.85-130.4). To conclude, genetic variants of COMT, MAOB and DRD2 loci modulate susceptibility to PD in South Indian subjects.
L-Asparaginase (L-asparagine amidohydrolase EC 18.104.22.168) from Erwinia aroideae NRRL B-138 has been purified to apparent homogeneity by ammonium sulphate precipitation, chromatography on sulfopropyl-sephadex C-50 and sephadex G-200 with 22% recovery and 567-fold purification. The enzyme obtained from sulfopropyl-sephadex C-50 was unstable and lost activity within a few hours. Addition of glycerol helped in restoring the activity of the enzyme. The enzyme has an apparent molecular mass of approximately 155 kDa and has four subunits of identical molecular mass of approximately 38 kDa. The K(m) for L-asparagine is 2.8 x 10(-3) M. Enzyme shows optimal activity at 45 degrees C and pH 8.2. Energy of activation as determined from Arrhenius plot was 9.1 kcal/mol. Substrate L-asparagine and analogue L-glutamine, D-asparagine and 6 diazo-5-oxo-L-norleucine provide full protection to the enzyme against thermal denaturation.
The results of the structural and conformational studies carried out using 13C CPMAS NMR technique on several glycine and alanine containing peptides in the solid state are reported. The study demonstrates the effects of variations in 13C chemical shifts due to conformation and hydrogen bonding. The possibility of applying this technique to obtain insight into the conformational characteristics of peptides of unknown structures is discussed.