Four cytosolic-type CuZn-superoxide dismutases in germinating seeds of Pinus sylvestris

A differential analysis of CuZn-superoxide dismutase (SOD. EC 1.15.1.1) isozymes after native-polyacry lamide gel elecrrophoresis (PAGE) and isoelectric focusing (IEF) indicated that germinating seeds of Scots pine (Pinus sylvestris L.) 3 days after the start of imbibition (3 DAI) contain five CuZn-SOD isozymes. Two isozymes co-migrated on native–PAGE but were separated after IEF. CuZn-SODs of Scots pine were purified from germinating seeds (3 DAI) by anion-exchange chromatography, hydrophobic interaction chromatography and chromatofocusing. The final separation of CuZn-SOD isozymes was accomplished by native-PAGE. CuZn-SOD isozymes were electroblotted and their NH₂-terminal amino acid sequence was determined. Comparisons of the amino acid sequences with sequences of CuZn-SOD isozymes from other plant sources indicated that one CuZn-SOD isozyme was of the chloroplastic type whereas the other four isozymes belonged to the cytosolic-type CuZn-SODs, The NH₂-terminal amino acid sequence of the chloroplastic CuZn-SOD and of one cytosolic-type CuZn-SOD were identical to those of two previously isolated, sequenced and localized CuZn-SOD isozymes from Scots pine needles. Two cytosolic-type CuZn-SOD isozymes showed a homology at 20 out of 21 NH₂-terminal amino acids. Mitochondria and glyoxysomes were isolated by differential and Percoll density-gradient centrifugation from germinating seeds (3 DAI). The cell fractionation experiments did not suggest that a major part of the CuZn-SOD activity in germinating seeds was derived from glyoxysomes or mitochondria.     

Novel Gemini vitamin D(3) analogs have potent antitumor activity

The active form of vitamin D(3), 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], modulates proliferation and induces differentiation of many cancer cells. A new class of analogs of vitamin D(3) has been synthesized, having two side-chains attached to carbon-20 (Gemini) and deuterium substituted on one side-chain. We have examined six of these analogs for their ability to inhibit growth of myeloid leukemia (HL-60), prostate (LNCaP, PC-3, DU145), lung (H520), colon (HT-29), and breast (MCF-7) cancer cell lines. Dose-response clonogenic studies showed that all six analogs had greater antiproliferative activities against cancer cells than 1,25(OH)(2)D(3). Although they had similar potency, the most active of these analogs was BXL-01-0120. BXL-01-0120 was 529-fold more potent than 1,25(OH)(2)D(3) in causing 50% clonal growth inhibition (ED(50)) of HL-60 cells. Pulse-exposure studies demonstrated that exposure to BXL-01-120 (10(-9)M, 48h) resulted in 85% clonal inhibition of HL-60 growth. BXL-01-0120 (10(-11)M, 4 days) induced the differentiation marker, CD11b. Also, morphologically differentiation was more prominent compared to 1,25(OH)(2)D(3). Annexin V assay showed that BXL-01-0120 (10(-10)M, 4 days) induced significantly (p<0.05) more apoptosis than 1,25(OH)(2)D(3). In summary, these analogs have a unique structure resulting in extremely potent inhibition of clonal proliferation of various types of cancer cells, especially HL-60 cells.     

Diversity of Selected Lupinus angustifolius L. Genotypes at the Phenotypic and DNA Level with Respect to Microscopic Seed Coat Structure and Thickness

The paper investigates seed coat characteristics (as a percentage of overall seed diameter) in Lupinus angustifolius L., a potential forage crop. In the study ten L. angustifolius genotypes, including three Polish cultivars, two Australian cultivars, three mutants originated from cv. ‘Emir’, and one Belarusian and one Australian breeding line were evaluated. The highest seed coat percentage was recorded in cultivars ‘Sonet’ and ‘Emir’. The lowest seed coat thickness percentage (below 20%) was noted for breeding lines 11257-19, LAG24 and cultivar ‘Zeus’ (17.87%, 18.91% 19.60%, respectively). Despite having low seed weight, the Australian line no. 11257-19 was characterized by a desirable proportion of seed coat to the weight of seeds. In general, estimation of the correlation coefficient indicated a tendency that larger seeds had thinner coats. Scanning Electron Microscopy images showed low variation of seed coat sculpture and the top of seeds covered with a cuticle. Most of the studied genotypes were characterized by a cristatepapillate seed coat surface, formed by elongated polygonal cells. Only breeding line no. 11267-19 had a different shape of the cells building the surface layer of the coat. In order to illustrate genetic diversity among the genotypes tested, 24 ISSR primers were used. They generated a total of 161 polymorphic amplification products in 10 evaluated narrow-leaved lupin genotypes.     

Serological characterization of anti-endotoxin serum directed against the conjugate of oligosaccharide core of Escherichia coli type R4 with tetanus toxoid

The covalent conjugate of oligosaccharide core of Escherichia coli type R4 with tetanus toxoid was prepared using reaction of reductive amination. The neoglycoconjugate was a good immunogen in rabbits yielding a high level of anti-lipopolysaccharide (LPS) antibodies of the IgG class. It was found that antiserum was able to react with the smooth LPS molecules of identical (R4) or related (R1) core type. The reactions were shown in the enzyme-linked immunosorbent assay and the immunoblotting test. Flow cytometry showed that anti-core antibodies reacted with LPS present on intact, live, smooth bacteria labelling more than 90% of cells. The anti-OS R4-TT serum used for in vitro studies showed high endotoxin neutralization activity. The serum inhibited endotoxin-induced tumor necrosis factor alpha and nitric oxide synthesis by the J-774A.1 cell line and attenuated pulmonary retention of YAC-1 cells.     

The road much traveled: trafficking in the cell nucleus

Trafficking of RNA molecules and proteins within the cell nucleus is central to genome function. Recent work has revealed the nature of RNA and protein motion within the nucleus and across the nuclear membrane. These studies have given insight into how molecules find their destinations within the nucleus and have uncovered some of the structural properties of the nuclear microenvironment. Control of RNA and protein trafficking is now emerging as a physiological regulatory mechanism in gene expression and nuclear function.     

Acetylcholinesterase: Role of the enzyme's charge distribution in steering charged ligands toward the active site

The electrostatic steering of charged ligands toward the active site of Torpedo californica acetylcholinesterase is investigated by Brownian dynamics simulations of wild type enzyme and several mutated forms, in which some normally charged residues are neutralized. The simulations reveal that the total ligand influx through a surface of 42 Å radius centered in the enzyme monomer and separated from the protein surface by I-14 Å is not significantly influenced by electrostatic interactions. Electrostatic effects are visible for encounters with a surface of 32 Å radius, which is partially hidden inside the protein, but mostly within the solvent. A clear accumulation of encounter events for that sphere is observed in the area directly above the entrance to the active site gorge. In this area, the encounter events are increased by 40% compared to the case of a neutral ligand. However, the differences among the encounter rates for the various mutants considered here are not pronounced, all rate constants being within ±10% of the average value. The enzyme charge distribution becomes more important as the charged ligand moves toward the bottom of the gorge, where the active site is located. We show that neither the enzyme's total charge, nor its dipole moment, fully account for the electrostatic steering of ligand to the active site. Higher moments of the enzyme's charge distribution are also important. However, for a series of mutations for which the direction of the enzyme dipole moment is constant within a few degrees, one observes a gradual decrease in the diffusional encounter rate constant with the number of neutralized residues. On the other hand, for other mutants that change the direction of the dipole moment from that of the wild type, the calculated encounter rate constants can be very close to that of the wild type. The present work yields two new insights to the kinetics of acetylcholinesterase. First, evolution appears to have built a redundant electrostatic steering capability into this important enzyme through the overall distribution of its thousands of partially charged atoms. And second, roughly half of the rate enhancement due to electrostatics arises from steering of the substrate outside the enzyme; the other half of the rate enhancement arises from improved trapping of the substrate after it has entered the gorge. The computational results reproduce qualitatively, and help to rationalize, many surprising experimental results obtained recently for human acetylcholinesterase. © 1996 John Wiley & Sons, Inc.     

Bifunctional indole-3-acetyl transferase catalyses synthesis and hydrolysis of indole-3-acetyl-myo-inositol in immature endosperm of Zea mays

1- O -(indole-3-acetyl)- β - d -glucose: myo -inositol indoleacetyl transferase (IA- myo -inositol synthase) is an important enzyme in IAA metabolism. This enzyme catalyses the transfer of the indole acetyl (IA) moiety from 1- O -(indole-3-acetyl)- β - d -glucose to myo -inositol to form IA- myo- inositol and glucose. IA- myo -inositol synthase was purified to an electrophoretically homogenous state from maize liquid endosperm by fractionation with ammonium sulphate, anion-exchange, adsorption on hydroxylapatite, affinity chromatography on ConA-Sepharose, preparative PAGE and isoelectric focusing. We thus obtained two enzyme preparations which differ in their R _f on 8% polyacrylamide gel. The preparation of R _f 0.36 contained a single 56.4 kDa polypeptide, whereas the preparation of R _f 0.39 consisted of two polypeptides of 56.4 and 53.5 kDa. Both purified preparations of IAInos synthase also exhibited the activity of an IAInos hydrolase, showing that the dual activity was associated with a single protein. Results of gel filtration and analytical SDS-PAGE suggest that the native enzyme exists as both a monomeric (65 kDa) and homo- or heterodimeric form (110–130 kDa). Analysis of peptide maps and amino acid sequences of two 21 amino-acid peptides showed that polypeptides of 56.4 and 53.5 kDa have the same primary structure and that the 3 kDa difference in molecular mass is probably caused by different glycosylation levels. Comparison of this partial and internal amino acid sequence with sequences of other plant acyltransferases indicated similarity to several proteins which belonged to the serine carboxypeptidase-like (SCPL) acyltransferase family.     

AutoMotif server: prediction of single residue post-translational modifications in proteins

The AutoMotif Server allows for identification of post-translational modification (PTM) sites in proteins based only on local sequence information. The local sequence preferences of short segments around PTM residues are described here as linear functional motifs (LFMs). Sequence models for all types of PTMs are trained by support vector machine on short-sequence fragments of proteins in the current release of Swiss-Prot database (phosphorylation by various protein kinases, sulfation, acetylation, methylation, amidation, etc.). The accuracy of the identification is estimated using the standard leave-one-out procedure. The sensitivities for all types of short LFMs are in the range of 70%. AVAILABILITY: The AutoMotif Server is available free for academic use at http://automotif.bioinfo.pl/