Evidence for a reduction of the replicative activity of matrix-bound DNA polymerase alpha following treatment with phospholipase C

Since phospholipids have been suggested to play some role in the molecular organisation of the nuclear matrix, the effect of their removal by means of phospholipase C has been investigated in regenerating rat liver nuclear matrix. The matrix-bound DNA polymerase alpha shows an almost complete loss of activity following the digestion with phospholipase C. Since the polymerase activity is restored by adding exogenous DNA, we suggest that the effect is due to the removal of matrix residual DNA, which is in some way linked to the nuclear matrix by means of hydrophobic interactions.     

Increased PARP-1 levels in nuclear matrix isolated from heat shock treated rat liver

Poly(ADP-ribose) polymerase-1 (PARP-1), a chromatin-associated enzyme that catalyzes the NAD⁺-dependent addition of ADP-ribose polymers onto a variety of nuclear proteins, has been shown to be associated with the nuclear matrix. PARP-1 levels in the nuclear matrix vary depending on the matrix isolation method used. The nuclear matrix appears to be the most thermosensitive nuclear structure during heat shock. Here we provide evidence for the extensive translocation of PARP-1 from chromatin to the nuclear matrix during heat shock. This translocation is accompanied by inhibition of PARP activity in the nucleus and elevation of PARP activity in the nuclear matrix. Our data suggest that thermal destabilization of the nuclear matrix is less likely to contribute to the translocation of PARP-1 to the nuclear matrix.     

Larvicidal activity of lectins from Myracrodruon urundeuva on Aedes aegypti

Aedes aegypti transmits etiologic agents of yellow fever and dengue. Vaccine for dengue virus is not available and vector control is essential to minimize dengue incidence. This report deals with the larvicidal activity of lectins isolated from Myracrodruon urundeuva bark (MuBL) and heartwood (MuHL). The lectins were isolated by ammonium sulphate treatment of crude extracts followed by chromatography on chitin. MuBL and MuHL were evaluated by electrophoresis under native (PAGE) and denaturing conditions (SDS-PAGE). Carbohydrate specificity of lectins was evaluated by hemagglutinating activity (HA) inhibition assay using N-acetyl-d-glucosamine and by affinity chromatography on N-acetyl-D-glucosamine immobilized in agarose gel. Larvicidal activity against A. aegypti was investigated with the extracts, salt fractions and isolated lectins. MuBL and MuHL were characterized by PAGE as basic proteins of molecular masses of 14.0 and 14.4 kDa, respectively. The interaction of lectins with N-acetylglucosamine was detected by inhibition of HA by monosaccharide and lectin adsorptions on N-acetyl-D-glucosamine matrix. All M. urundeuva preparations promoted larvae mortality. LC16, LC50 and LC84 values of 0.077, 0.125, 0.173 for MuBL and 0.03, 0.04 and 0.05 mg/mL for MuHL were obtained. To our knowledge this is the first report of larvicidal activity of lectins against A. aegypti.     

An isolectin complex from Trichosanthes anguina seeds

Sepharose 4B affinity chromatography of Trichosanthes anguina seed extract and subsequent elution with galactose resulted in the isolation of an apparently single lectin with molecular weight of 45,000 +/- 700. However, major amount of the hemagglutinating activity was recovered as unadsorbed protein fraction. High affinity matrix Lactamyl Seralose could retain most of the galactose specific lectin activity from fraction 'A' which was eluted with lactose. It is evident from PAGE and SDS-PAGE analysis of the purified protein that T. anguina seeds contains a mixture of isolectins ranging in molecular weight from 30,000 to 50,000 +/- 1300. Periodic Acid Schiff's staining of the gels revealed this lectin complex to be a combination of glycosylated and non-glycosylated lectins. Two Isolectins SLc and IEL from within this complex have been isolated by affinity and ion exchange chromatography respectively. Apparent homology of these two lectins is indicated by their identical molecular weight (45 kDa), sub unit composition, non glycoprotein nature and immunological identity. However, these two lectins show minor differences in their biological and physicochemical properties. The peptide maps of the two lectins obtained after digestion with Trypsin and Pronase E also indicate minor changes in the primary structure.     

ADP-ribosylation intensifies cleavage of DNA loops in the nuclear matrix

Using DNA pulse field electrophoresis it has been shown that ADP-ribosylation in the nucleoids of human mononuclear leukocytes and rat brain cortex neurons stimulates cleavage of DNA loops at their attachmant sites to the nuclear matrix. The conclusion has been drawn suggesting possible participation of ADP-ribosylation in DNA-topoisomerase II activity modulation in the nuclear matrix of eukaryotic cells.     

Participation of Poly(ADP-ribose)-polymerase of nuclear matrix in DNA repair

The poly(ADP-ribose)-polymerase activity of brain and liver cell nuclei is changed during X-irradiation of rats. In the nuclear matrix, poly(ADP-ribose)-polymerase activity increases at a low dose of irradiation (1.7 Gy) and decreases at a high dose (6.7 Gy). A significant part of the activity of nuclear NMN-adenylyltransferase, a key enzyme for biosynthesis of NAD (the substrate of poly(ADP-ribose)-polymerase), has been found in the nuclear matrix. An interrelation between ADP-ribosylation taking place on the matrix level and eukaryotic cell DNA repair is suggested.     

Neoglycoenzymes: a versatile tool for lectin detection in solid-phase assays and glycohistochemistry

Carbodiimide-mediated coupling of p-aminophenyl glycosides to a naturally nonglycosylated enzyme yields a neoglycoenzyme. This compound combines inherent enzymatic activity with synthetically conferred ligand properties to lectins. Appropriate choice of the ligand allows custom-made synthesis to reliably detect various types of lectins. To exemplify practical applications of this class of compounds, glycosylated bacterial beta-galactosidase has been employed to quantitate plant lectins, immobilized on plastic surfaces as well as on nitrocellulose. Competitive inhibition by specific sugar ascertained the dependence of binding on protein--carbohydrate interactions. In view of lectins as tools, a sandwich lectin-binding assay for high mannose-type glycoprotein detection has been modified to principally facilitate wide application to other lectin-reactive sugar chains by introducing the neoglycoenzyme. In addition to lectin determination in solid-phase assays, neoglycoenzymes allow one to glycohistochemically localize endogenous lectins in tissue prints and tissue sections with a minimum number of steps. This nonradioactive, rapid, sensitive, and convenient assay concept, based on conjugation of a ligand to an enzyme with maintenance of its receptor-binding activity, may find extended application beyond lectinology in receptor analysis.     

The identification of inducible cytoplasmic/nuclear carbohydrate-binding proteins urges to develop novel concepts about the role of plant lectins

During the last few years compelling evidence has been presented for the occurrence of cytoplasmic/nuclear plant lectins that are not detectable in normal plants but are only induced upon application of well-defined stress conditions. Since both the regulation of the expression and the subcellular location indicate that these ‘non-classical lectins’ are good candidates to play a physiologically important role as mediators of specific protein-carbohydrate-interactions within the plant cell, a critical assessment is made of the impact of these findings on the development of novel concepts about the role of plant lectins. Based on an analysis of the biochemical, molecular and evolutionary data of a jasmonate-induced chitin-binding lectin from tobacco leaves and a salt/jasmonate-induced leaf lectin from rice it is concluded that these lectins most probably interact with endogenous glycans located within the cytoplasmic/nuclear compartment of the plant cell. Several working mechanisms are proposed to explain how these inducible lectins may fulfill an important regulatory or structural role in stressed cells. In addition, the question of the evolutionary relationship(s) between the newly discovered inducible lectins and their ‘classical’ constitutively expressed homologs is addressed. Evidence is presented that the ‘non-classical lectins’ represent the main evolutionary line and that some of their corresponding genes were used as templates for genes encoding storage protein-like ‘classical’ homologs. Published in 2004.     

Distribution of lectins in membranes of soybean and peanut plants

The distribution of lectin activity in soybean and peanut plants has been investigated. In both plants activity is found in all tissues examined (roots, shoots and leaves) at all stages of development from seedling to maturity (7 weeks). The cellular location of the lectins differs between soybean and peanut: in soybean the lectins are generally membrane-associated, whereas in peanut plants lectin activity is present also in the soluble cytoplasmic fraction. The membrane-associated lectins appear to differ from the seed lectins of the respective plants. The function of membrane-associated lectins is discussed.Abbreviations RCA lectin of castor bean - SBA soybean agglutinin - PNA peanut agglutinin - HEPES 2-[4-(2-Hydroxyethyl)-piperazinyl-(1)]ethanesulphonic acid - MES morpholinoethane sulphonic acid - PBS phosphate-buffered saline     

Purification and characterization of a beta-galactoside-binding soluble lectin from rat and bovine brain

A beta-galactoside-binding activity has been detected in mammalian brain extracts using a hemagglutination test and a nerve cell aggregation assay. Inhibition studies suggested the involvement of lectin-carbohydrate interactions in these processes. In an attempt to explore further the biological role of brain lectins, the beta-galactoside-binding activity has been purified to apparent homogeneity from bovine and rat brain by salt extraction of the brain tissue and affinity chromatography on asialofetuin-agarose. The molecular weights determined by gel filtration, under native conditions on Ultrogel AcA-34, were 30,000 for the bovine brain lectin and 32,000 for the rat brain lectin; polyacrylamide gel electrophoresis in SDS gave molecular weights of 15,000 and 16,000, respectively, suggesting that the two brain lectins are dimers. Both lectins have an isoelectric point of 3.9. Amino acid composition data indicate that both lectins contain high proportions of glycine and acidic amino acids. The lectins are specific for beta-D-galactosides and related sugars and the configuration of carbon atoms 1, 2 and 4 seems of primary importance. Moreover, the nerve cell aggregation-promoting activity of the purified lectin is 300-fold that of the crude extracts.     

Protease activity of the nuclear matrix of rat hepatocytes

It was demonstrated that the nuclear matrix of rat liver possesses the protease activity. The specific activity of nuclear matrix proteases exceeds that of intact nuclei 7-fold. The optimum activity of nuclear matrix proteases is observed at pH 8-9. The protease activity of the nuclear matrix is inhibited by p-chloromercuribenzoate, N-ethylmaleimide, EDTA, phenylmethylsulfonyl fluoride. This suggests that thiol, serine and metalloproteases are associated with the nuclear matrix.     

An assay for leukoagglutinating lectins using suspension cultured mouse lymphoma cells (BW5147) stained with neutral red

A sensitive and rapid assay for leukoagglutinating lectins has been developed. This assay utilizes neutral red-stained mouse lymphoma cells from the suspension cultured cell line BW5147. The agglutination of the stained cells can be monitored visually in a manner similar to that for conventional assays for erythroagglutinating lectins using erythrocytes. The activity of lekoagglutinating lectins that are not capable of agglutinating erythrocytes can be quantified by this assay. The utility of the assay was demonstrated using leukoagglutinating and erythroagglutinating lectins from the seeds of Phaseolus vulgaris and Maackia amurensis.     

DNA-polymerase activity and DNA synthesis in the hepatocyte nuclear matrix

The comparative analysis of DNA-synthetase activity of hepatocytes, isolated nuclei and nuclear matrix from normal and regenerating rat liver was performed. The highest enrichment with newly-synthesized DNA was registered in the DNA fraction associated with the nuclear matrix both in vivo and in vitro. The functioning of DNA polymerases alpha and beta in the matrix was shown. Our results indicate that DNA polymerase beta is more firmly bound with the nuclear matrix in the cells of normal liver but this enzyme is eluted almost completely from the nuclei of regenerating liver cells. At the first moment after gamma-irradiation of rats the preferential initiation of unscheduled DNA synthesis in vivo has been observed on the nuclear skeletal structures. This may serve as an indication on the possibility that DNA repair process occurs on the nuclear matrix.     

Inhibition by cysteine of the carbohydrate-binding activity of lectins from Ricinus communis, Canavalia ensiformis and Euonymus europaeus

Precipitation induced by different lectins has been studied in the presence of some aminoacids. It was shown that precipitates formed by lectins from Ricinus communis (RCA1), Canavalia ensiformis (Con A), Euonymus europaeus (Eel) in the presence of appropriate carbohydrate-containing molecules disappeared after cysteine addition, like after addition of specific carbohydrate precipitation inhibitors. It is assumed that cysteine residues of RCA1, Con A and Eel lectins are essential for their carbohydrate binding activity.     

Structure-function relationships amongLathyrus lectins

The structure-function relationship of eight structurally very similarLathyrus lectins, which are two-chain lectins from theVicieae tribe, has been studied. Hemagglutination activity, inhibition of hemagglutination, glycoprotein reactivity and mitogenic activity were determined for each of the lectins. Despite the close structural resemblance, marked differences in the carbohydrate binding activity of theLathyrus lectins were recorded. These functional differences were related to small conformational changes due to minor differences in amino acid sequence of eachLathyrus lectin.     

A histochemical study of the distribution of lectin binding sites in the developing oocytes of the lancelet Branchiostoma belcheri

The distribution of carbohydrate moieties in lancelet (Branchiostoma belcheri) oocytes has been studied at different stages of development, using a peroxidase-labeled lectin incubation technique, the PAS-reaction and Alcian Blue staining. Binding sites of 5 lectins, indicating the presence of different sugar moieties (Wheat germ agglutinin (WGA) for N-acetylglucosamine, Concanavalin A (Con A) for glucose/mannose, Helix pomatia agglutinin (HPA) for N-acetyl-D-galactosamine, Ricinus communis agglutinin (RCA-I) for galactose and Ulex europaeus agglutinin (UEA-I) for fucose), were identified and were shown to undergo considerable variation during oocyte development. In the previtellogenic stage, HPA, RCA-I and UEA-I were not identified on the oocyte surface, but WGA and Con A gave strongly positive reactions at this site. In the cytoplasm, 4 lectins (Con A, HPA, RCA-I and UEA-I) gave a weak or moderate reaction, and Con A was also observed in the perinuclear region. In vitellogenic oocytes, these 4 lectins were found to also bind to the nuclear envelope, karyoplasm and nucleolus, and, with the exception of Con A, could also be found in the nuclei of more mature stages. The cytoplasmic yolk granules and Golgi vesicles of the vitellogenic oocyte, were moderately positive for Con A, HPA, RCA-I and UEA-I, but HPA, RCA-I and UEA-I were only weakly bound at the oocyte surface. In mature oocytes, all 5 lectins bound moderately or strongly to yolk granules and cell surface. HPA, RCA-I and UEA-I bound moderately or strongly to various nuclear compartments. Thus, carbohydrate content varied with the development and maturation of the oocytes, and the PAS results were in agreement with the lectin-binding results. Charged carbohydrate residues were observed in the egg envelope and Golgi bodies.These results suggest that the appearence of Con A-, HPA-, RCA-I- and UEA-I-binding glycoconjugates in the nuclei of developing oocytes show a varying pattern indicating different phases of nuclear activity which correlate with different carbohydrate synthetic activities of the oocyte.     

A dot blot technique for the analysis of interactions of lectins with glycosaminoglycans

Summary Glycosaminoglycans are polysaccharides which are widely distributed throughout connective tissue, where they form an essential part of the extracellular matrix. Connective tissue is often stained by lectins, and it is not known whether this staining is due to the interaction of lectins with the glycosaminoglycans or due to the binding of lectins to other glycoconjugates within the matrix. A dot blot technique is presented by which the interaction of lectins with glycosaminoglycans can be analysed.     

Spatial conformation of glycans and glycoproteins

Ten years ago, we anticipated future results by building the Y-shaped molecular model of a biantennary glycan. Progressively, this structure has been refined and modified thanks to experimental data obtained by using physical methods: X-ray diffraction, electron spin resonance (EPR), nuclear magnetic resonance (NMR) including two-dimensional NMR and one-dimensional 1H-nuclear Overhauser effect (NOE) experiments, neutron scattering and hard-sphere exo-anomeric (HSEA) calculations. So, the concept evolved successively from the Y-, to the T-, the bird- and the "broken wing"-conformation, until the demonstration, that these conformers are interconvertible. The bird-conformation as well as the concept of the mobility of antennae are in a good agreement with the reactivity of lectins, including membrane lectins, by rendering accessible any specific sugar structure, and with the activity of glycosyltransferases by making reachable the substitutable hydroxyl groups even in the case of pentaantennary structures. Along this line, we know now that the tetraantennary glycans adopt an "umbrella conformation" in which the four antennae are disposed parallely to the protein surface and act as protective shields. So could be explained the resistance towards proteases and the weak antigenicity of numerous glycoproteins as well as the peculiar behaviour and resistance of metastatic cancerous cells since it has been recently demonstrated that membrane glycoproteins and fibronectin of this kind of cells are significantly enriched in tri- and tetraantennary glycans.     

Affinity-repulsion chromatography. Principle and application to lectins

The interactions of proteins with their immobilized ligands in an electrically charged microenvironment were studied. The binding of lectins to erythrocytes and to affinity matrices was used as a model system. Lectins bind and agglutinate erythrocytes in the presence of at least 10 mM NaCl or 1 mM CaCl2, but not in deionized water. The salt dependence of the agglutination process is due to the ability of salts to provide counterions neutralizing the forces of repulsion between the electrostatic charges of similar sign present on the erythrocyte cell surface and on the lectins. The same salt dependence is observed for the binding of lectins to affinity matrices. These observations are the basis of a protein separation process coined affinity-repulsion chromatography in which the electrostatic charges present, or purposely introduced, on affinity matrices are exploited and allow the elution, by electrostatic repulsion, of proteins carrying electrostatic charges of the same sign as that of the matrix. In this process, proteins are loaded on the affinity matrix in a salt solution and eluted with deionized water. Affinity-repulsion chromatography has been successfully applied here to the isolation of several lectins. Its physicochemical basis, merits, and potential applications are discussed.