The effect of specific glycosidases on Ricinus communis agglutinin binding to cell surfaces of two tumor sublines. A comparative flow-cytometric study

The effects of the sequential application of specific glycosidases on surfaces of living mammalian cells were studied with respect to their ability to bind the beta-galactoside-specific lectin, Ricinus communis agglutinin (RCA). Sialidase and beta-galactosidases from different sources were tested for their actions on two strains of mouse lymphoma cells differing markedly in their metastatic potential. Binding studies were performed by quantitative flow cytometry with fluorescent RCA, and numbers of specific binding sites and equilibrium association constants for the lectin on living cells were determined before and after the various enzyme treatments. Although the number of binding sites for native and sialidase-treated cells were almost identical for both cell strains, differences in the apparent affinity constants could be detected. Differences between the two strains became even more pronounced, also with respect to the number of binding sites, after treatment with beta-galactosidases from S. pneumoniae and from bovine testis. It is suggested that such combined strategies provide valuable tools for the differentiation of surface carbohydrate moieties on intact living cells, especially for comparative purposes.     

The localization and the isoenzymes of α- and β-Galactosidases in root tips

The localization was studied of α- and β-galactosidases in frozen sections of Ca-formol fixed root tips using simultaneous azocoupling reaction. In all species studied (Allium cepa,Cucurbita maxima, Lupinus albus, Pisum sativum, Vicia faba, Zea mays) positive results were obtained, the localization being ubiquitous (according to localization typology given here). InVicia faba andZea mays the isoenzymes of α- and β-galactosidases were revealed by means of acrylamide gel electrophoresis, using authors’ modification of Reisfeld method, in whole root tips, particular growth zones and separately in cortex and central cylinder. No differences were observed comparing stele and cortex. Whereas characteristic isoenzyme patterns were found in individual growth zones in maize, no differences appeared in broad bean. A comparison was made of thein situ localization and of the isoenzyme patterns of α- and β-galactosidases with α- and β-glucosidases. In the case of galactosidases, positive results appear with both α- and β-galactoside. The rising of pH to neutrality leads to considerable decrease in the activity of both galactosidases.     

Progressive induction of β-glucuronidase in individual kidney epithelial cells

The magnitude and kinetics of β-glucuronidase induction in mouse kidney are determined by a cis-acting regulatory gene, Gus-r, that is closely linked to the enzyme structural gene. The accumulation of β-glucuronidase mRNA during induction is much slower than the turnover time of the mRNA, suggesting progressive acquisition of mRNA synthesizing capacity during induction. Counts of the numbers of induced cells present at various times of induction in strains carrying three different alleles of Gus-r show that all potentially responsive cells respond immediately. The level of induction is progressive in individual cells and does not involve continued recruitment of new cells into the induced population. It appears that during induction each chromosome becomes progressively more active in directing the synthesis of β-glucuronidase.     

Segregation of genetic determinants for murine glucuroinidase synthesis and loss in CXB recombinant-inbred strains

A set of recombinant-inbred strains developed from mouse strains BALB/c and C57BL/6 includes two β-glucuronidase phenotypes that are not seen in either of the progenitor strains. These new recombinant phenotypes indicate that glucuronidase levels are regulated by genes additional to the Gur locus, which is closely linked to the glucuronidase structural gene (Gus) and is known to regulate the rate of glucuronidase synthesis. In this study, induced rates of glucuronidase synthesis were determined for these recombinant-inbred strains, and rate constants for enzyme loss were calculated. The rate of synthesis was found to segregate with the Gus gene in all of the strains, and only the determinants for rate of enzyme loss recombined to give new phenotypes. It was concluded that at least two genes affect the rate of enzyme loss, that these genes are not closely linked to each other or to the Gur-Gus region on chromosome 5, and that no major determinants of glucuronidase synthesis segregate independently of Gur.     

Insights into the effects of glycosylation and the monosaccharide-binding activity of the plant lectin CrataBL

CrataBL is a glycoprotein isolated from Crataeva tapia bark, containing two N-glycosylation sites. It has been identified to present lectin activity with some specificity for binding glucose over galactose. However, to date, no information on the effects of glycosylation or CrataBL monosaccharide-binding sites and monosaccharide specificity has been obtained. Thus, molecular docking and molecular dynamics simulations were employed to characterize the glycosylated CrataBL conformation and dynamics in aqueous solutions, as well as the molecular basis for its binding specificity. The obtained results indicate both local and distant conformational stabilization effects of N-linked glycans over CrataBL protein moiety. Regarding its lectin activity, molecular docking calculations were performed in two possible binding sites, identified through sequence-based, structure-based and evolutionary information, using α- and β-anomeric states of the monosaccharides. The obtained poses were further refined through molecular dynamics simulations, suggesting that positively-charged amino acids dictate the binding preference for glucose over galactose in both sites. In addition, a possible preference for β-monosaccharides was proposed. Such data are expected to contribute to a better comprehension of the lectins monosaccharide-binding activities and carbohydrate-binding site structures.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either ¹²⁵I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10⁷ binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37°C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.     

Further characterization of α-galactosidase I-glycoprotein lectin from Vicia faba seeds

The nature of the glucose/mannose specific lectin activity of α-galactosidase I from Vicia faba seeds has been examined. Gel filtration in the presence of high concentrations of glucose and SDS-PAGE failed to detect favin, a classical lectin which also occurs in the seed. A comparison of the haemagglutinating activities of the α-galactosidases from Vigna radiata and V. faba seeds strongly suggests that the catalytic site of the Vigna enzyme is also responsible for its agglutinating activity and that the catalytic and lectin sites are at different loci in the case of V. faba α-galactosidase I. The latter conclusion is supported by an investigation of the effects of glucose, mannose and galactose on the catalytic and lectin activities and by results obtained by demetallization of the V. faba enzyme. A single galactose-binding site and two mannose binding sites per subunit of enzyme I were detected by the method of equilibrium dialysis and the association constants for these monosaccharides measured. Mannose did not appear to affect the binding of galactose to the enzyme or vice versa. The removal of glycan chains from α-galactosidase I with endo-β-N-acetylglucosaminidase H released an active dimeric form of α-galactosidase. The possible involvement of lectin-glycoprotein interactions in the stabilization of the tetrameric form of the enzyme is considered.     

Antimicrobial and Antibiofilm Activity of Mauriporin,a Multifunctional Scorpion Venom Peptide

Many pathogenic free living and biofilm forming bacterial organisms can cause serious infections to humans that could consequently have devastating effects on human health. A significant number of these microbial organisms are resistant to almost all known conventional antibiotics and the ability of some these strains to form sessile communities of biofilms increases the resistance ability of bacteria to antibiotic treatment. Global research is currently focused on finding novel therapies to counteract the threat of bacterial and biofilm infections rather than using conventional antibiotics. Mauriporin, a novel cationic α-helical peptide identified from the venom derived cDNA library of the scorpion Androctonus mauritanicus was reported to display selective cytotoxic and anti-proliferative activity against prostate cancer cell lines. In the present study, we investigated the antimicrobial and antibiofilm activities of Mauriporin. Our results show that Mauriporin displays potent antimicrobial activities against a range of Gram-positive and Gram-negative planktonic bacteria with MIC values in the range 5 µM to 10 µM. Mauriporin was also able to prevent Pseudomonas aeruginosa biofilm formation while showing weak hemolytic activity towards human erythrocytes. Studies on the mechanism of action of Mauriporin revealed that the peptide is probably inducing bacterial cell death through membrane permeabilization determined by the release of β-galactosidase enzyme from peptide treated Escherichia coli cells. Moreover, DNA binding studies found that Mauriporin can cause potent binding to intracellular DNA. All these results indicate that Mauriporin has a considerable potential for therapeutic application as a novel drug candidate for eradicating bacterial infections.     

Assessment of ecological diversity of rhizobacterial communities in vermicompost and analysis of their potential to improve plant growth

Present study was designed to determine the microbial diversity from three distinctive sites (amended with vermicompost) of Gujarat, India. A set of 76 strains were screened from total of 438 strains that exhibit plant growthpromoting (PGP) and antagonistic potential isolated from sites PS1 (Mehsana district), BS2 (Dantiwada district) and VS3 (Gandhinagar district). Their diversity indices were studied for determining the species richness and evenness of screened isolates. Results revealed that site BS2 showed the most significant diversity indices in terms of Shannon (H′ 1.525) and Simpson (1/D 5.120) than other two samples. Principal component analysis showed that bacterial diversity (H′) was correlated with the soil characteristics. Chickpea and groundnut plants inoculated with MBCU1 and MBCU3 isolates showed an increase in the vegetative growth parameters that evaluate plant growth when compared to uninoculated controls. Strains MBCU1 and MBCU3 were identified as Pseudomonas stutzeri and Pseudomonas mosselii, respectively, according to sequence analysis of the 16S rRNA gene. These both isolates belong to site BS2 and they showed specific PGP traits suggesting that these isolates can promote plant growth by more than one mechanism with respect to their higher diversity index.     

Interaction of synthetic glycopeptides carrying clusters ofO-glycosidic disaccharide chains (β-d-Gal(1–3)-α-d-GalNAc) withβ-d-galactose-binding lectins

The specificity of severald-galactose-binding lectins including Agaricus bisporus (mushroom),Arachis hypogaea (peanut),Bauhinia purpurea andVicia graminea has been examined by inhibition of hemagglutination using a series of synthetic oligopeptides representing the N-terminal end of glycophorin A from N and M individuals, all carrying one or several disaccharide chains,d-Galβ1–3-d-GalNAcα-(T-hapten). Peanut lectin was inhibited by T-hapten-carrying glycopeptides, but the presence of a cluster of disaccharide chains had no effect on the lectin specificity. On the contrary, bothAgaricus bisporus andBauhinia purpurea lectins exhibited an enhanced reactivity with polyglycosylated peptides suggesting that their combining site might include two proximal galactose residues. All synthetic glycopeptides inhibitingVicia graminea lectin carry a cluster of T-disaccharide chains and the leucine residue at the N-terminal end, and the presence of a Glu residue at position 5 slightly increased the lectin activity. It is concluded that the binding ofVicia graminea is dependent upon a specific spatial conformation including a cluster of T-hapten chains in close vicinity of a hydrophobic surface represented by an appropriate N-terminal amino acid residue.     

Lectin binding sites in normal and phenobarbitale/halothane treated rat liver

Summary The content of carbohydrate residues of both normal and phenobarbitale-halothane-hypoxia exposed rat liver has been examined by means of lectin histochemistry. Eight biotinylated lectins specific to galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, fucose and mannose were applied to paraffin sections of rat liver at light microscopic levels. The most distinct binding was observed at the structures of the perisinusoidal functional unit: Kupffer cells are bound by S-WGA, SBA and PNA. Bile canaliculi display binding sites for RCA I and WGA. Cytoplasm of hepatocytes appears lectin-negative, except for PSA. The enhanced reaction of S-WGA, PNA and SBA after the preincubation of the sections with neuraminidase indicates the occurance of sialic acid in Kupffer cells. The phenobarbitale-halothane-hypoxia exposed rat liver shows centrolobular degeneration of hepatocytes with a diminished amount of hepatocytes and Kupffer cells as well. The lectin binding pattern of sinusoidal walls, membranes of hepatocytes and bile canaliculi remains the same compared to that of normal rat liver. This finding suggests that at least the carbohydrate content of membranes in the liver resists severe destruction under phenobarbitale-halothane-hypoxia. It is assumed that there exists a connection between intact carbohydrate residues and the regeneration of liver parenchyma.     

Decrease in human lymphocyte surface glycoconjugates in leukemia as demonstrated by lectin binding

Qualitative variations in the glycoconjugates which make up the lectin receptor sites on the membranes of leukemic lymphocytes, compared with those of normal cells, have been studied by the use of three tritiated lectins: Robinia pseudoacacia lectin, Concanavalin A and Ricinus communis (var. Sanquineus) agglutinin (RCA 120). The binding specificity of these lectins has been demonstrated using specific determinants: alpha-methylmannoside and galactose for Concanavalin A and Ricinus communis agglutinin respectively. For the Robinia lectin this specificity was determined by saturation of the receptor sites with the unlabeled Robinia lectin before the addition of isotopically labeled Robinia lectin. The results show a decrease in the number of receptor sites on the leukemia cells, especially in chronic lymphoid leukemia, relative to that on normal cells. The apparent affinity constants of leukemic cells in all cases remain higher than those of normal cells.     

Bacterial community analysis in chlorpyrifos enrichment cultures via DGGE and use of bacterial consortium for CP biodegradation

The organophosphate pesticide chlorpyrifos (CP) has been used extensively since the 1960s for insect control. However, its toxic effects on mammals and persistence in environment necessitate its removal from contaminated sites, biodegradation studies of CP-degrading microbes are therefore of immense importance. Samples from a Pakistani agricultural soil with an extensive history of CP application were used to prepare enrichment cultures using CP as sole carbon source for bacterial community analysis and isolation of CP metabolizing bacteria. Bacterial community analysis (denaturing gradient gel electrophoresis) revealed that the dominant genera enriched under these conditions were Pseudomonas, Acinetobacter and Stenotrophomonas, along with lower numbers of Sphingomonas, Agrobacterium and Burkholderia. Furthermore, it revealed that members of Bacteroidetes, Firmicutes, α- and γ-Proteobacteria and Actinobacteria were present at initial steps of enrichment whereas β-Proteobacteria appeared in later steps and only Proteobacteria were selected by enrichment culturing. However, when CP-degrading strains were isolated from this enrichment culture, the most active organisms were strains of Acinetobacter calcoaceticus, Pseudomonas mendocina and Pseudomonas aeruginosa. These strains degraded 6–7.4 mg L^?1 day^?1 of CP when cultivated in mineral medium, while the consortium of all four strains degraded 9.2 mg L^?1 day^?1 of CP (100 mg L^?1). Addition of glucose as an additional C source increased the degradation capacity by 8–14 %. After inoculation of contaminated soil with CP (200 mg kg^?1) disappearance rates were 3.83–4.30 mg kg^?1 day^?1 for individual strains and 4.76 mg kg^?1 day^?1 for the consortium. These results indicate that these organisms are involved in the degradation of CP in soil and represent valuable candidates for in situ bioremediation of contaminated soils and waters.     

Population ecology of Sulfolobus acidocaldarius

Ecology of Sulfolobus acidocaldarius was studied in situ by the use of the immunofluorescence and immunodiffusion techniques. The fluorescent antibodies (FA) prepared against four strains of Sulfolobus were highly reactive against their homologous antigens. Two of the FA's were strain specific and the other two exhibited reciprocal corssreactions against each other's antigens, but immunodiffusion patterns showed that the two strains were not identical. The growth of a serologically distinct isolate in a hot spring was measured by immunofluorescence staining of immersion slides. On glass immersion slides Sulfolobus grew and formed colonies with a mean-doubling time of approximately 36 h. Immunofluorescence was applied to study the geographical distribution of two serologically different strains and to establish population composition of individual springs. One strain was found in all sites studied, and most springs contained more than one serologic type. Immunodiffusion was capable of detecting specific Sulfolobus antigens in hot springs which contained a high population of FA-reactive cells.     

Haemagglutinin inTriticale

Saline extracts of several varieties ofTriticale had haemagglutinin activity against rabbit, rat and fowl erythrocytes. In contrast to the wheat germ lectin theTriticale lectin was inactive against human B, 0 blood group type erythrocytes and rather high concentrations of the lectin are needed to agglutinate human A blood group type erythrocytes. TheTriticale lectin was purified about 20-fold with a 10% recovery of activity from one of the varieties (DTS 138) by (NH₄)₂SO₄ fractionation followed sequentially by chromatography on DEAE-cellulose and sulphopropyl-Sephadex. Approximately 4 μg of the purified lectin caused visible agglutination with trypsinised rabbit erythrocytes. Among a variety of sugars tested D-glucose, D-mannose and N-acetyl-D-glucosamine (2·5-7·5mM) caused inhibition of agglutination.     

Copper tolerance in Frankia sp. strain EuI1c involves surface binding and copper transport

Several Frankia strains have been shown to be copper-tolerant. The mechanism of their copper tolerance was investigated for Frankia sp. strain EuI1c. Copper binding was shown by binding studies. Unusual globular structures were observed on the surface of the bacterium. These globular structures were composed of aggregates containing many relatively smaller “leaf-like” structures. Scanning electron microscopy with energy-dispersive X-ray (SEM-EDAX) analysis of these structures indicated elevated copper and phosphate levels compared to the control cells. Fourier transform infrared spectroscopy (FTIR) analysis indicated an increase in extracellular phosphate on the cell surface of copper-stressed cells. Bioinformatics’ analysis of the Frankia sp. strain EuI1c genome revealed five potential cop genes: copA, copZ, copC, copCD, and copD. Experiments with Frankia sp. strain EuI1c using qRT-PCR indicated an increase in messenger RNA (mRNA) levels of the five cop genes upon Cu²⁺ stress. After 5 days of Cu²⁺ stress, the copA, copZ, copC, copCD, and copD mRNA levels increased 25-, 8-, 18-, 18-, and 25-fold, respectively. The protein profile of Cu²⁺-stressed Frankia sp. strain EuI1c cells revealed the upregulation of a 36.7 kDa protein that was identified as FraEuI1c_1092 (sulfate-binding periplasmic transport protein). Homologues of this gene were only present in the genomes of the Cu²⁺-resistant Frankia strains (EuI1c, DC12, and CN3). These data indicate that copper tolerance by Frankia sp. strain EuI1c involved the binding of copper to the cell surface and transport proteins.