Crystallization and preliminary X-ray diffraction analysis of PD-L4, a ribosome inactivating protein from Phytolacca dioica L. leaves

PD-L4, a type 1 ribosome inactivating protein from Phytolacca dioica leaves, has been successfully crystallized using vapour diffusion methods and PEG 4000 as a precipitant agent. In addition, crystals of a PD-L4 mutant, which has been recently observed to have a lower polynucleotide-adenosine glycosidase activity on DNA, rRNA and poly (A) substrates, have been obtained. To gather information on PD-L4 reaction mechanism both forms have been co-crystallized with adenine, the major product of their catalytic reaction. Diffraction patterns extend to atomic resolution and crystals belong to the orthorhombic P2(1)2(1)2(1) space group, with one molecule in the asymmetric unit. Structure determination has been achieved using molecular replacement; preliminary electron density maps have clearly given evidence of adenine binding.     

Assay for Laccase activity by microcalorimetry: laccase was extracted from china lacquer of Rhus vernicifera

The reactions between Laccase (extracted from China lacquer of Rhus vernicifera) and various substrates (3,4-Dihydroxybenzaldehyde, Guaiacol, Pyrogallol, Gallic acid) have been studied using LKB-2107 batch microcalorimetry system. Based on calorimetry, a new method has been proposed. Laccase activity and the Michaelis constant K(m) have been determined simultaneously by this method. The method is simple, sample-saving, and valid for a wider range of substrate concentrations. Furthermore, it can be extended for assaying other enzymes catalyzing reactions using this method.     

QSAR studies on benzene sulfonamide carbonic anhydrase inhibitors: need of hydrophobic parameter for topological modeling of binding constants of sulfonamides to human CA-II

The binding constants (logK) of benzene sulfonamides to human CA-II have been modeled using a large series of distance-based topological indices. The need or otherwise of the hydrophobic parameter (logP) for such topological modeling of logK has been examined critically. In both the cases excellent results have been obtained. In multiparametric models involving indicator parameters we observed that introduction of hydrophobic parameter (logP) yields much improved statistics. The results are discussed on the basis of statistical parameters and also by using cross-validation method.     

A Study of Second Order Approximation of Some Ratio Type Strategies

For estimating the mean of a finite population using information on an auxiliary variable, the conventional ratio strategies and strategies due to Srivastava (1967), Reddy (1973), Gupta (1978), Sahai (1979) and Adhvaryu-Gupta (1983) have been studied. Asymptotic expressions for the second order approximations of biases and mean square errors of these strategies have been obtained. The suitability of these strategies have been discussed with the help of live data.     

Flanking sequence tags in Arabidopsis thaliana T-DNA insertion lines: a pilot study

Eight hundred and fifty Arabidopsis thaliana T-DNA insertion lines have been selected on a phenotypic basis. The T-DNA flanking sequences (FST) have been isolated using a PCR amplification procedure and sequenced. Seven hundred plant DNA sequences have been obtained revealing a T-DNA insertion in, or in the immediate vicinity of 482 annotated genes. Limited deletions of plant DNA have been observed at the site of insertion of T-DNA as well as in its left (LB) and right (RB) T-DNA signal sequences. The distribution of the T-DNA insertions along the chromosomes shows that they are essentially absent from the centrometric and pericentrometric regions.     

Synthesis of MRI contrast agents derived from DOTAM-Gly-l-Phe-OH incorporating a disulfide bridge: Conjugation to a cell penetrating peptide and preparation of a dimeric agent

A cell penetrating peptide conjugate and dimeric PARACEST MRI contrast agents, based on the DOTAM-Gly-l-Phe-OH scaffold have been prepared in moderate yields using diethyl azodicarboxylate (DEAD) or iodine-mediated disulfide bridge formation as a key step. Magnetic (PARACEST) properties of these agents have been evaluated.     

Antiradical and NO-inhibiting activities of bet-hydroxy(ethoxy) derivatives of nitrous heterocycles

The antiradical and NO-inhibiting activities of beta-hydroxy(ethoxy) derivatives of nitrous heterocycles (3-hydroxypyridine, 5-hydroxybenzimodazole, and 6-hydroxy(alkoxy)-benzothiazole) have been studied. The antiradical activity has been studied using a homogeneous hydrophilic chemiluminescent system, and the quenching constants (Ki) have been determined. For the most reactive compound, 4-methylthiobenzimidazolyl-3-hydroxypyridine, Ki = 4.5 x 105 M(-1). The NO-inhibiting activity was estimated on a model of the endotoxin shock of experimental animals using a spin trap of nitric oxide radicals based on complexes of iron with sodium diethyldithiocarbamate. It was found that the compounds at doses of 0.25-1 mmol/kg have both the inhibitory and stimulating action on the production of nitric oxide in the liver of animals. The results obtained suggest that some derivatives of nitrous heterocycles can be used as effective antioxidant preparations.     

Quantification of DNA structure from NMR data: conformation of d-ACATCGATGT

Resonance assignments of nonexchangeable base and sugar protons have been obtained in double-helical d-ACATCGATGT by using two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The exchangeable imino protons have been assigned on the basis of their chemical shifts. The characteristic phase-sensitive multiplet patterns of the intrasugar cross-peaks in the omega 1-scaled COSY spectrum have been used to estimate several scalar coupling constants (J). The information on the J values combined with the intranucleotide COSY cross-peak intensities has been used to identify sugar puckers of individual nucleotide units. In most cases, the deoxyribofuranose rings are found to adopt a conformation close to O4'-endo. Spin diffusion has been monitored from the buildup of the normalized volumes of NOE cross-peaks in NOESY spectra as a function of mixing time. A set of 52 intranucleotide and internucleotide proton-proton distances have been estimated by using low mixing time NOESY spectra (tau m = 40 and 80 ms). The estimated intrasugar proton-proton distances rule out possibilities of existence of a fast equilibrium between C2'-endo and C3'-endo conformations. Intranucleotide proton-proton distances combined with the knowledge of sugar puckers have been used to fix the glycosidic bond torsion angle (chi). For this purpose, simulated distance contours depicting the dependence of intranucleotide proton-proton distances on pseudorotational phase angle (P) and glycosidic bond torsion angle (chi) have been used. Further, the proton homonuclear (J, delta) spectrum has been used to monitor the 31P-1H heteronuclear couplings, which are preserved in the omega 2 projection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plant mutant tubulin genes as marker selective genes for genetic engineering

The approaches for new marker genes usage in selection of transformed plant cells, which are based on using mutant tubulin genes from natural plant biotypes and, in perspective, from induced plant mutants have been considered. The results of investigations of plant (biotypes, mutants) resistance to herbicides with antimicrotubular mode of action on molecular and cellular levels have been summarized. The reports dealing with study the transferring and the expression of mutant tubulin genes conferring resistance to amiprophosmethyl (phosphorothioamidate herbicide) and to trifluralin (dinitroaniline herbicide) from corresponding N. plumbaginifolia mutants into related and remote plant species by somatic hybridisation methods have been analyzed. The results of experiments on monocotyledonous and dicotyledonous. plant transformation by mutant alpha-tubulin gene conferring resistance to dinitroanilines are described to test the possibility of its using as a marker gene with obtaining, at the same time, a dinitroaniline-resistant plants.     

Aluminum Nanohole Arrays Fabricated on Polycarbonate for Compact Disc-Based Label-Free Optical Biosensing

Al nanohole array plasmonic biosensors have been fabricated on polycarbonate (PC) substrates from conventional compact discs (CD). Standard micro and nanofabrication processes have been used and optimized to be PC compatible. The viability of this CD-based plasmonic platform for label-free optical biosensing has been demonstrated through a competitive bioassay for biotin analysis using biotin-functionalized dextran-lipase conjugates immobilized on the transducer surface.     

Zinc complexes with cyclic derivatives of alpha-ketoglutaric acid thiosemicarbazone: Synthesis, X-ray structures and DNA interactions

Six new cyclic ligands derived from alpha-ketoglutaric acid thiosemicarbazone (H(3)ct) and their zinc complexes have been synthesized and characterized by analytical and spectroscopic (IR and NMR) studies. The X-ray structures of ligands Me-H(2)ct(C) (1), Allyl-H(2)ct(c) (3) and of complex [Zn(Me-Hct(C))(2)(OH(2))(2)].2H(2)O (7) have been determined. In complex (7) the zinc atom lies on a twofold axis and is surrounded in a tetrahedral coordination by two water molecules and two carboxylic oxygen donor atoms from the ligand. DNA titration in the UV-visible region and thermal denaturation have been employed to determine the details of DNA binding for the studied compounds. Studies of nuclease activity have also been performed with all our compounds through a gel electrophoresis experiment using plasmid pBR322 showing that no DNA breakings take place. Tests in vitro on human leukemia cell line U937 have been carried out on cell growth inhibition where solubility of the compounds allowed the experiments.     

Herbivory and plant resource competition: a review of two interacting interactions

This review discusses the prevalence and potential for interactive effects between herbivory and competition on plant growth and biomass, and it is apparent that such effects typically arise when there is a mismatch between the spatial scale of herbivore behaviour (food or patch choice) and the spatial heterogeneity of the plant community. Historically, such interactive effects have been examined using two approaches. Studies using the first approach have excluded plant neighbors and herbivores in a factorial experiment, and scored effects on plant biomass. Studies using the second approach have observed herbivore abundance or herbivory on plants with or without plant neighbors, and have identified a large number of mechanisms underlying such interactive effects. The two types of studies have produced somewhat conflicting results, where interactive effects have been commonly observed in studies using the second approach and only rarely in studies using the first approach. This is most likely a consequence of a biased choice of study systems, where studies using the first approach have primarily studied mammalian herbivory while studies using the second approach have been more focussed on insect herbivory. Moreover, studies using the first approach have typically been very small-scale manipulations and this probably precludes most possible interactive effects in systems with mammalian herbivory. This points to the fact that studies examining interactive effects of herbivory and plant competition should more carefully consider the behaviour and life history of herbivores included in the study prior to the design of removal experiments.     

Bioactive apocarotenoids from Tectona grandis

The bioactive fractions of Tectona grandis have yielded seven apocarotenoids, two of which have been isolated for the first time as natural products (tectoionols A and B). The chemical structures were determined through 1D and 2D nuclear magnetic resonance experiments. The absolute configuration of tectoionol A was determined using a modified Mosher methodology. Some NMR assignments for the compounds 9(S)-4-oxo-7,8-dihydro-beta-ionol and 3beta-hydroxy-7,8-dihydro-beta-ionone have been corrected on the basis of g-HSQC and g-HMBC experiments. The general bioactivities of isolated compounds have been studied using etiolated wheat coleoptiles. Those compounds that presented higher levels of activity were assayed on standard target species (Lactuca sativa, Lycopersicum esculentum, Lepidium sativum and Allium cepa).     

A physiologically based approach for degree-day calculation in pest phenology models: the case of the European Corn Borer (<Emphasis Type="BoldItalic">Ostrinia nubilalis</Emphasis> Hbn.) in Northern Italy

Phenological models based on degree-day accumulation have been developed to support the integrated pest management of many insects. Most of these models are based on linear relationships between temperature and development, and on daily time step simulations using daily minimum and maximum temperatures. This approach represents an approximation that does not take into account the insect physiological response to temperature, and daily temperature fluctuations. The objective of this work has been to develop a phenological model for the European corn borer (ECB) based on the insect physiological response to temperature and running at an hourly time step. Two modeling solutions based on the same generic compartmental system have been compared: the first based on a physiologically based relationship between temperature and development, and using hourly derived temperatures as input (HNL modeling solution); and the second based on a linear relationship between temperature and degree-day accumulation and using daily temperature (DL modeling solution). The two approaches have been compared using ECB moth capture data from the Piemonte region in Northern Italy. The HNL modeling solution showed the best results for all the accuracy indicators. The DL modeling solution showed a tendency to anticipate ECB phenological development too early. This tendency is attributable to the linear relationship between temperature and development, which does not take into account (1) the decline of this relationship at high temperatures, and (2) the daily fluctuation of temperature. As a consequence, degree-days accumulation is accelerated in the DL modeling solution and the phenological development anticipated.     

Bone mineral density and microarchitecture in participants of a 105-day experiment in isolated environment

A study on the bone system state in healthy volunteers has been performed before and after 105-day experiment in hermetically isolated environment (the Mars-105 experiment) using dual energy X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT). The values of bone mineral density (BMD), volumetric bone mineral density (VBMD), and bone structural characteristics of distal segments in radius and tibia have been evaluated. No significant DXA changes have been revealed in segments of skeleton critically important in terms of biomechanics. Microarchitectural deterioration (a decrease in the trabecula number and increase in the bone tissue heterogeneity) has been found using the pQCT technique in the radius of the majority of subjects. A VBMD decrease has been revealed for both cortical and trabecular bones in tibia, along with an unexpected trabecular bone improvement in the form of an increase in the trabecula quantity and decrease in bone tissue heterogeneity. Comprehensive studies, including estimation of projective and volumetric bone mineral densities (the bone mineral content) and bone structural characteristics (bone quality) are required to have a clear view on the changes in the bone system under the conditions of a simulation experiment.     

QSAR study on 1-benzyl-4[w(substituted phthalimido)alkyl]piperidines:acetyl cholinesterase inhibitors.

QSAR studies on a series of 18 piperidine derivatives, which act as acetyl cholinesterase (AchE) inhibitors, have been performed using van der Waals volume (V omega) and topochemical index (tau). Significant correlations have been obtained, which make it clear that AchE inhibition activity is controlled dominantly by topo chemical index.     

Development of a highly sensitive ELISA for the determination of PBAN and its application to the analysis of hemolymph in Spodoptera littoralis

A highly sensitive enzyme linked immunosorbent assay (ELISA) for the determination of the pheromone biosynthesis activating neuropeptide (PBAN) has been developed. Six antisera have been obtained that recognize the carboxyl terminal side of this peptide. Two immunogens have been rationally designed and synthesized in order to direct antibody specificity, using as haptens PBAN or PBAN(20-33) with a Cys residue attached to their amino-terminal side. The Cys thiol group has been used to covalently bind the peptide to keyhole limpet hemocyanin (KLH) by using N-succinimidyl-4-(maleidimidomethyl) cyclohexane carboxylate (SMCC) as a convenient heterobifunctional cross-linker. Several usable competitive immunoassays have been obtained by synthesizing eight different coating antigens and screening the sera against all of them. The best assay was obtained with antibody 4 using Cys-Hez-PBAN(20-33) coupled to bovine serum albumin (BSA) through the Lys groups by using the homobifunctional cross-linker dimethylpimelidate dihydrochloride (DMP) as the coating antigen. The optimized assay allows to detect PBAN at concentrations as low as 1 fmol/well (l₅₀ = 2.5 fmol/well). An extraction procedure for the hemolymph has been developed that allows to perform PBAN measurements in this tissue even after a tenfold dilution. In these conditions matrix effect is negligible. Preliminary results on the presence of PBAN like immunoreactivity (PBAN-IR) in the hemolymph of Spodoptera littoralis females are reported.© 1995 Wiley-Liss, Inc.     

Solid-phase modification with succinic polyethyleneglycol of aminated lipase B from Candida antarctica: Effect of the immobilization protocol on enzyme catalytic properties

Lipase B from Candida antarctica (CALB) has been modified using succinic polyethyleneglycol via the carbodiimide route. Immobilized enzyme (on octyl Sepharose or Eupergit C) has been used, to take advantage of the solid phase. Modification of immobilized CALB's native amino groups did not produce a significant alteration of CALB. However, if the enzyme was previously aminated, around 14–15 PEG molecules could be introduced per enzyme molecule. Also, it has been found that succinic groups are far more reactive than acetic acid following this strategy.Even after this drastic double modification, the functional properties of the enzyme have not been impoverished to a large extent: stability decreased only to some extent (by a 5–6 fold factor), activity versus some substrates even increased (e.g., around 60% using p-nitrophenyl butyrate). It has been found that both modifications (amination and pegylation) have very different effects on enzyme properties when performed on CALB immobilized on Eupergit C or octyl Sepharose. For example, activity versus pNPP increased using CALB-octyl Sepharose while it decreased when using Eupergit C following amination and PEGylation. The effects also depend on the reaction and substrate, for example in hydrolysis of methyl mandelate, the activity decreased by 50% using CALB-octyl Sepharose after PEGylation of the aminated enzyme, while using CALB-Eupergit C had no effect. In this last case, enantioselecitvity in this hydrolysis significantly improved after both chemical modifications (from 7.5 to 20), while using CALB-octyl Sepharose almost had no effect.     

Structure-activity relationship studies of a series of peptidomimetic ligands for alpha(4) beta(1) integrin on Jurkat T-leukemia cells

alpha(4)beta(1) integrin is a therapeutic target for inflammation, autoimmune diseases, and lymphoid cancers. A series of peptidomimetic ligands based on the Nle-D-I motif have been synthesized and their binding affinities (IC(50)) to activated alpha(4)beta(1) integrin on Jurkat T-leukemia cells have been determined using a cell adhesion assay. One of the 51 ligands, 18, has been determined to have an IC(50) of 0.6 nM and has a more than twofold increase of binding affinity than the initial lead compound 1. Extensive SAR studies provide important information for further ligand optimization, which has served as a foundation for studies that ultimately led to identification of a potent ligand with an IC(50) of 2 pM.     

Ultrastructural studies of peripheral blood lymphocytes in T cell-depleted rabbits

Summary Density separation of purified peripheral blood leucocytes from T-cell depleted rabbits on a linear Ficoll-metrizoate gradient has been applied to obtain different leucocyte fractions. Two lymphocyte fractions separated on density seem to have different characteristics, both morphologically and immunologically. In this study these two fractions have been characterized ultrastructurally by using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and a relationship has been established between the surface architecture (SEM), the cell size (SEM/TEM) and surface-Ig/C₃-receptors (LM, light microscopy). Finally three types of lymphocytes have been described in the two lymphocyte fractions separated on density. Morphometric information such as cell size, cell shape, eu-/heterochromatin ratio in the nucleus and the nucleus-/cell ratio have been correlated to the stage of activation of the B lymphocyte in a representative density separation.