Effects of subinhibitory concentrations of antibiotics on biological properties ofSalmonella typhimurium

We followed the effects of subinhibitory concentrations (sub-MICs) of 7 antibiotics (ticarcilin, cefotaxim, streptomycin, gentamicin, ciprofloxacin, pefloxacin, mitomycin C) on the sensitivity of aSalmonella typhimurium strain to standard bacteriophages, on the phage DNA as well as on the factors of virulence (permeability and cytotoxic activity). The phage type was not changed by the sub-MICs of the tested antibiotics. However, differences were found in culture filtrates prepared from the bacterial suspensions of the strain cultivated with the sub-MICs. Marked inducing effects on phage DNA were exhibited by mitomycin C (1/2, 1/4, 1/8 of the MIC), pefloxacin (1/2, 1/4, 1/8 of the MIC) and ciprofloxacin (1/2, 1/4, weakly also 1/8 of the MIC). Ticarcilin (1/2 of the MIC), like the aminoglycosides streptomycin and gentamicin (1/2, 1/4, 1/8 of the MIC), had a weak effect. Sub-MICs of the studied antibiotics (with the exception of 1/8 of the MIC of ciprofloxacin and 1/4 of the MIC of ticarcilin) decreased the permeability reaction in rabbit skin. Most effective was streptomycin (1/2 of the MIC). Sub-MICs of the tested antibiotics (with the exception of 1/4 and 1/8 of the MIC of ciprofloxacin and 1/4 of the MIC of pefloxacin) caused also an inhibition of the factor responsible for morphological changes on Vero cells. Gentamicin and streptomycin were effective at all the sub-MICs tested.     

The insecticidal activity of recombinant garlic lectins towards aphids

The heterodimeric and homodimeric garlic lectins ASAI and ASAII were produced as recombinant proteins in the yeast Pichia pastoris. The proteins were purified as functional dimeric lectins, but underwent post-translational proteolysis. Recombinant ASAII was a single homogenous polypeptide which had undergone C-terminal processing similar to that occurring in planta. The recombinant ASAI was glycosylated and subject to variable and heterogenous proteolysis. Both lectins showed insecticidal effects when fed to pea aphids (Acyrthosiphon pisum) in artificial diet, ASAII being more toxic than ASAI at the same concentration. Acute toxicity (mortality at 3d exposure) was observed over the concentration range 0.125-2.0mgml(-1). The recombinant lectins caused mortality in both symbiotic and antibiotic-treated aphids, showing that toxicity is not dependent on the presence of the bacterial symbiont (Buchnera aphidicola), or on interaction with symbiont proteins, such as the previously identified lectin "receptor" symbionin. A pull-down assay coupled with peptide mass fingerprinting identified two abundant membrane-associated aphid gut proteins, alanyl aminopeptidase N and sucrase, as "receptors" for lectin binding.     

Externalization of the endogenous intracellular lectin of a cellular slime mold

Intracellular purpurin, the endogenous lectin of Dictyostelium purpureum, has previously been shown to be externalized upon exposure of the cells to anti-purpurin IgG. Externalization of additional purpurin was presumably the consequence of cross-linking of the purpurin molecules already on the cell surface by the IgG, since binding univalent anti-purpurin Fab to the cell surface did not have this effect. In the present report we show that multivalent glycoconjugates that interact with purpurin—including asialo-bovine submaxillary mucin, a bacterial galactan, and bovine albumin derivatized with multiple chains of either lactose or lacto-N-neotetraose—all elicit the externalization of intracellular purpurin. Some of the externalized lectin is bound to the cell surface and some is found in the medium, presumably bound to the glycoconjugates. A similar effect is produced by exposure of the cells to high concentrations of purified purpurin. Several plant lectins, including conA, also have some effect, whereas others are inactive. Since cells in late stages of aggregation have about three times as much cell surface purpurin as those in early stages, this externalization reaction may have significance late in development. The results suggest that intracellular purpurin may be released in response to cross-linking of the endogenous cell surface glycoconjugates that are: (1) already bound to endogenous purpurin; (2) capable of binding purpurin; (3) capable of binding certain other lectins. The intracellular lectin may be a reserve pool, that functions only upon externalization.     

Peptidoglycan recognition proteins kill bacteria by activating protein-sensing two-component systems

Mammalian peptidoglycan recognition proteins (PGRPs), similar to antimicrobial lectins, bind the bacterial cell wall and kill bacteria through an unknown mechanism. We show that PGRPs enter the Gram-positive cell wall at the site of daughter cell separation during cell division. In Bacillus subtilis, PGRPs activate the CssR-CssS two-component system that detects and disposes of misfolded proteins that are usually exported out of bacterial cells. This activation results in membrane depolarization, cessation of intracellular peptidoglycan, protein, RNA and DNA synthesis, and production of hydroxyl radicals, which are responsible for bacterial death. PGRPs also bind the outer membrane of Escherichia coli and activate the functionally homologous CpxA-CpxR two-component system, which kills the bacteria. We exclude other potential bactericidal mechanisms, including inhibition of extracellular peptidoglycan synthesis, hydrolysis of peptidoglycan and membrane permeabilization. Thus, we reveal a previously unknown mechanism by which innate immunity proteins that bind the cell wall or outer membrane exploit the bacterial stress defense response to kill bacteria.     

Distribution of lectin during the life cycle of Phaseolus vulgaris L.

Phaseolus vulgaris L. cv. ‘garrafal encarnada’ plants have been utilized to study the distribution of lectins accumulated in the seeds and through the life cycle of the plant.The distribution of both, total proteins and lectins was studied in aqueous and saline (1 M NaCl) extracts from different parts and organs in four distinct stages of plant development.Our results showed that lectin concentration decreases sharply during the first weeks of the plant growth, reaching the lowest value in trifoliate leaf stage and increasing during the following phases of plant development. However, the presence of lectins have been detected in all the plant tissues through every phase of the life cycle.The observed differences on lectin levels (RIA) and lectin activity (hemagglutination), suggest the presence of different molecular forms of the lectin in aqueous and saline extracts of plant tissues.These results, as well as the observation about the fixation of lectin on the bacterial surface, support the idea that the function of lectins in the plant may not be limited to storage proteins, but may be involved in specific host-parasite recognition.     

Receptors of Garlic (Allium sativum) Lectins and Their Role in Insecticidal Action

Garlic (Allium sativum) lectins are promising candidate molecules for the protection against chewing (lepidopteran) as well as sap sucking (homopteran) insect pests. Molecular mechanism of toxicity and interaction of lectins with midgut receptor proteins has been described in many reports. Lectins show its effect right from sensory receptors of mouth parts by disrupting the membrane integrity and food detection ability. Subsequently, enter into the gut lumen and interact with midgut glycosylated proteins like alkaline phosphatase (ALP), aminopeptidase-N (APN), cadherin-like proteins, polycalins, sucrase, symbionin and others. These proteins play critical role in life cycle of insect directly or indirectly. Lectins interfere with the activity of these proteins and causes physiological disorders leading to the death of insects. Lectins further transported across the insect gut, accumulated in various body parts (like haemolymph and ovary) and interact with intracellular proteins like symbionin and cytochrome p450. Binding with cytochrome p450 (which involve in ecdysone synthesis) might interfere in the development of insects, which results in growth retardation and pre-mature death.     

Cross-linking of streptomycin to the 50S subunit of Escherichia coli with phenyldiglyoxal

[3H]Dihydrostreptomycin was covalently linked to the 50S subunit of Escherichia coli K12A19 with the bifunctional cross-linking reagent phenyldiglyoxal. The cross-linking was abolished under conditions that prevent the specific interaction of streptomycin with the ribosome. The binding primarily involved the ribosomal RNA and also a limited number of proteins, namely, L2, L6, and L17. This suggests that the binding domain for streptomycin is close to the peptidyl transferase center, in the valley between the central protuberance and the wider lateral protuberance of the 50S subunit. This domain faces the binding domain for streptomycin which we have previously characterized on the 30S subunit [Melan?on, P., Boileau, G., & Brakier-Gingras, L. (1984) Biochemistry 23, 6697-6703]. Our results indicate that the 50S subunit is involved in the binding of streptomycin to the bacterial ribosome, in addition to the 30S subunit which is generally considered as the specific target of the antibiotic. They are consistent with the occurrence of a single binding site for streptomycin on the ribosome, comprised of regions of both subunits.     

Role of the endothelial glycocalyx in dromotropic, inotropic, and arrythmogenic effects of coronary flow

Coronary flow regulates cardiac functions, and it has been suggested that endothelial membrane glycosylated proteins are the primary shear stress mechanosensors. Our hypothesis was that if these proteins are the sensors for flow, then intracoronary perfusion of lectins or specific antibodies should differentially depress coronary flow-enhanced responses of different parenchymal cell types such as auricular-ventricular (A-V) nodal cells (dromotropic effect), contractile myocytes (inotropic effect), and junctional Purkinje-muscle cells (spontaneous ventricular rhythm). The coronary flow stimulatory effects on A-V delay and spontaneous ventricular rhythm were selectively depressed by six of eight lectins. None of the lectins depressed the coronary flow inotropic effect. Antibodies against endothelial surface proteins, alpha(v)beta(5)-integrin and sialyl-Lewis(b) glycan, depressed the dromotropic but not the inotropic effects of coronary flow, whereas the vascular cell adhesion molecule 1 antibody had no effect on the dromotropic, but enhanced the inotropic, effect. The fact that lectins and antibodies differentially depressed regional coronary flow effects suggests that there is a chemical distinctiveness in their intravascular endothelial cell surfaces. However, nonselective cross-linking of endothelial glycocalyx proteins with 2,000-kDa dextran-aldehyde or vitronectin indistinctively depressed the dromotropic and inotropic effects of coronary flow. These results indicate that coronary flow-induced stress acts on specific structures located in the capillary intravascular membrane glycocalyx.     

Mitogen stimulation of T-cells increases c-Fos and c-Jun protein levels, AP-1 binding and AP-1 transcriptional activity.

We have analysed the effect of mitogenic lectins on c-Fos and c-Jun protein levels as well as on activator protein-1 (AP-1) binding and enhancer activity in Jurkat T-cells. Both c-Fos and c-Jun protein levels were increased after Con A and PHA stimulation. Since T-cell stimulation increases both intracellular Ca2+ and cAMP levels and activates protein kinase C (PKC), the possible involvement of these intracellular messengers in c-Fos and c-Jun induction was tested. PMA, which directly activates PKC, mimicked the effect of the lectins on c-Fos and c-Jun, but elevation of either intracellular Ca2+ or cAMP levels had little or no effect. The mitogen-induced increase of c-Fos and c-Jun immunoreactivity was inhibited by H-7, a kinase inhibitor with relatively high specificity for PKC, and less efficiently by H-8, a structurally related kinase inhibitor less active on PKC, but more active on cyclic nucleotide-dependent kinases. Con A stimulation was found to increase both binding of AP-1 to the AP-1 consensus sequence, TRE, and AP-1 enhancer activity, in Jurkat cells. PMA was also found to increase the AP-1 enhancer activity, whereas elevation of Ca2+ or cAMP had only minor effects. We conclude that stimulation with mitogenic lectins is sufficient to increase both c-Fos and c-Jun protein levels, AP-1 binding and AP-1 enhancer activity in Jurkat cells and that they act via mechanisms that could involve the activation of PKC.     

Isolation of specific RNA-binding proteins using the streptomycin-binding RNA aptamer

Here we report a simple and cheap one-step affinity purification protocol for isolating RNAs or proteins that interact with selected functional RNAs. The streptomycin-binding aptamer, termed 'StreptoTag,' is embedded in or fused to either end of any RNA of interest. The resulting hybrid RNA can then be immobilized on a streptomycin affinity matrix. When a complex protein mixture or total cellular lysate is applied to the matrix, subsequent elution with free streptomycin allows efficient recovery of specific ribonucleoprotein or RNA-RNA complexes. The method was successfully used to purify yeast and phage RNA-binding proteins and group II intron, viral and bacterial noncoding RNA (ncRNA)-binding proteins. The selective enrichment of bacterial mRNAs that bind ncRNAs has also been demonstrated. Once the affinity matrix, the RNA construct and the protein extracts have been prepared, the experimental procedure can be performed in 1-2 h.     

Lectin-like proteins in model organisms: implications for evolution of carbohydrate-binding activity.

Classes of intracellular lectins that recognize core-type structures and mediate intracellular glycoprotein trafficking are present in vertebrates, model invertebrates such as Caenorhabditis elegans and Drosophila melanogaster, plants, and yeasts. Lectins that recognize more complex structures at the cell surface, such as C-type lectins and galectins, are also found in invertebrate organisms as well as vertebrates, but the functions of these proteins have evolved differently in different animal lineages.     

Lectins of the <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> pollen grain walls stimulating in vitro pollen germination

Proteins diffusing from tobacco pollen grains into external medium, being inactivated by low temperature (0°C), were shown to stimulate pollen germination in vitro. Fractionation of these proteins by affinity chromatography using α-D-methylmannopyranoside (MMP) immobilized on agarose resulted in the isolation of lectins stimulating germination. The mol wts of these lectins were estimated by SDS-PAGE as 58, 69, and 74 kD. A stimulatory effect of these lectins was determined by their specific interaction with carbohydrate determinants because a competitive sugar (0.3 M MMP) suppressed completely lectin effect on germination. Polyvalent lectins capable of erythrocyte agglutination were also found among diffused proteins. These lectins are glycoproteins with Glu/Man carbohydrate determinants. MMP did not affect their capability of agglutination. This finding permits a conclusion that pollen grain wall contains lectins differing in their carbohydrate specificity.     

Streptomycin-induced conformational changes in the 70-S bacterial ribosome.

Conformational alterations induced by streptomycin in the bacterial ribosome have been investigated using as probes, ethidium bromide, N-[14C]ethylmaleimide and a spin label nitroxide analog of N-ethylmaleimide. 1. The binding of the antibiotic to the ribosome does not affect the reactivity of sulfhydryl groups towards N-ethylmaleimide. 2. The motional freedom of spin labels bound to ribosomal proteins S1 and S18 is increased but it is hardly affected at other labeled sites. This observation suggests that the binding of streptomycin causes a local loosening of the ribosomal structure. 3. Ribosomes are found to bind less ethidium bromide in the presence of streptomycin, which suggests that the binding of streptomycin decreases the degree of organization of ribosomal RNA.     

The X-lectins: A new family with homology to the Xenopus laevis oocyte lectin XL-35

The Xenopus laevis oocyte cortical granule lectin (XL35) has been studied in fertilization and embryonic development. Several nucleic acid sequences that predict proteins homologous to XL35 have since been reported in frog, human, mouse, lamprey, trout, ascidian worm. These proteins also showed high degrees of amino acid sequence homology to a common fibrinogen-like motif that may involve carbohydrate binding. Although their biological functions and carbohydrate binding specificities have not been studied in detail, this new family of lectins has common characteristics. Several independent studies on this new family of lectins strongly suggest that the lectins are expressed and stored in specialized vesicles that may be released upon the infection by pathogens. In addition, some family members have been shown to bind to oligosaccharides from bacterial pathogens. Therefore, this family of lectins likely participates in pathogen surveillance as part of the innate immune system. We propose the name X-lectin family for these homologs of XL35. Published in 2004.     

Effects of phytohaemagglutinin, wheat-germ agglutinin, and concanavalin-A on the physical state of sialic acid and membrane proteins in human erythrocyte ghosts: a spin label study

Parallel experiments employing sialic acid- and protein specific spin labels have been performed to monitor the effects on the physical state of this carbohydrate and membrane proteins of human erythrocytes induced by the binding of three lectins, $\text{Phaseolus vulgaris}$ phytohaemagglutinin (PHA), wheat germ agglutinin (WGA), and Concanavalin-A (Con-A). PHA and WGA, both of which are known to bind at different sites on the principal sialoglycoprotein of human erythrocytes, glycophorin, had markedly different effects: compared to control values, PHA decreased the apparent correlation time of the sialic acid specific spin probe by 10% while this parameter was decreased by 33% by WGA. The protein specific spin label also monitored differential effects of these lectins: the relevant electron spin resonance parameter (the W/S ratio) was reduced 33% by PHA and increased by WGA over 17% from that of control values. Con-A, which is known to bind to the principal transmembrane protein, Band 3, had no effect on sialic acid or membrane proteins as assessed by the two spin labels employed. These results suggest that (1) the effects of binding of these different lectins, two of which bind to the same cell surface receptor, can be discriminated by use of spin labeling methods; (2) binding events occuring at the cell surface have distinct and pronounced effects on the physical state of proteins within the membrane; (3) the different results with PHA and WGA both of which bind to glycophorin are indicative of multiple and complex interactions of this glycoprotein with the membrane proteins in the erythrocyte; and (4) that the spin labelling technique has the potential to investigate the relationships between cell-surface binding events to membrane structural-functional interactions.     

Uptake by macrophages of a biotinylated oligo-alpha-deoxythymidylate by using mannosylated streptavidin.

Streptavidin substituted with mannose residues increased by 20-fold the intracellular concentration of a biotinylated dodecakis(alpha-deoxythymidylate) in macrophages by comparison with the uptake of free oligodeoxynucleotide. Streptavidin, the bacterial homologue of the very basic avidin, which does not contain any carbohydrate moieties and is a neutral protein, was substituted with 12 mannose residues in order to be recognized and internalized by mannose-specific lectins on the surface of macrophages. A 3'-biotinylated and 5'-fluoresceinylated dodecakis (alpha-deoxythymidylate) was synthesized and bound onto mannosylated streptavidin. The conjugate was isolated, and by using flow cytometry, it was shown that the uptake of fluoresceinylated oligodeoxynucleotides bound to mannosylated streptavidin by macrophages is 20-fold higher than that of free oligodeoxynucleotides and that the uptake was competively inhibited by mannosylated serum albumin. Glycosylated streptavidin conjugates recognizing specific membrane lectins on different cells provide the possibility to target biotinylated antisense oligodeoxynucleotides and to increase the biological effect of these chemotherapeutic agents.     

Glycans as endocytosis signals: the cases of the asialoglycoprotein and hyaluronan/chondroitin sulfate receptors

Animal cells internalize specific extracellular macromolecules (ligands) by using specialized cell surface receptors that operate through a complex and highly regulated process known as receptor-mediated endocytosis, which involves the binding, internalization, and transfer of ligands through a series of distinct intracellular compartments. For the uptake of a variety of carbohydrate-containing macromolecules, such as glycoproteins, animal cells use specialized membrane-bound lectins as endocytic receptors that recognize different sugar residues or carbohydrate structures present on various ligands. The hepatic asialoglycoprotein receptor, which recognizes glycoconjugates containing terminal galactose or N-acetylgalactosamine residues, was the first membrane lectin discovered and has been a classical system for studying receptor-mediated endocytosis. Studies of how the asialoglycoprotein receptor functions have led to the discovery of two functionally distinct, parallel pathways of clathrin-mediated endocytosis (called the State 1 and State 2 pathways), which may also be utilized by all the other endocytic recycling receptor systems. Another endocytic membrane lectin, the hyaluronan/chondroitin sulfate receptor, which has recently been purified and cloned, is responsible for the turnover in mammals of these glycosaminoglycans, which are important components of extracellular matrices. We discuss the characteristics and physiological importance of these two proteins as examples of how lectins can function as endocytic receptors.     

Genomic analysis of C-type lectins

Many biological effects of complex carbohydrates are mediated by lectins that contain discrete carbohydrate-recognition domains. At least seven structurally distinct families of carbohydrate-recognition domains are found in lectins that are involved in intracellular trafficking, cell adhesion, cell-cell signalling, glycoprotein turnover and innate immunity. Genome-wide analysis of potential carbohydrate-binding domains is now possible. Two classes of intracellular lectins involved in glycoprotein trafficking are present in yeast, model invertebrates and vertebrates, and two other classes are present in vertebrates only. At the cell surface, calcium-dependent (C-type) lectins and galectins are found in model invertebrates and vertebrates, but not in yeast; immunoglobulin superfamily (I-type) lectins are only found in vertebrates. The evolutionary appearance of different classes of sugar-binding protein modules parallels a development towards more complex oligosaccharides that provide increased opportunities for specific recognition phenomena. An overall picture of the lectins present in humans can now be proposed. Based on our knowledge of the structures of several of the C-type carbohydrate-recognition domains, it is possible to suggest ligand-binding activity that may be associated with novel C-type lectin-like domains identified in a systematic screen of the human genome. Further analysis of the sequences of proteins containing these domains can be used as a basis for proposing potential biological functions.     

Polyamines and the Accumulation of Ribonucleic Acid in Some Polyauxotrophic Strains of Escherichia coli

The cellular accumulations of polyamines and ribonucleic acid (RNA) were compared in the polyauxotrophic mutants of Escherichia coli strain 15 TAU and E. coli K-12 RC(re1) met(-) leu(-). Putrescine, spermidine, and their monoacetyl derivatives were the main polyamines in both strains, when grown in glucose-mineral medium. No significant degradation of either (14)C-putrescine or (14)C-spermidine was found in growing cultures of strain 15 TAU, which requires thymine, arginine, and uracil for growth. Experiments with this organism showed that in a variety of different incubation conditions, which included normal growth, amino acid starvation, inhibition by chloramphenicol or streptomycin, or thymine deprivation, a close correlation was seen between the intracellular accumulation of unconjugated spermidine and RNA. In the presence of arginine, the antibiotics stimulated the production of putrescine and spermidine per unit of bacterial mass. Deprivation of arginine also resulted in an increase in the production of putrescine per unit of bacterial mass, most of which was excreted into the growth medium. However, in this system the antibiotics reduced the synthesis of putrescine. Furthermore, streptomycin caused a rapid loss of cellular putrescine into the medium. The latter effect was not seen in anaerobic conditions or in a streptomycin-resistant mutant of 15 TAU. Methionine added to the growth medium of growing TAU not only markedly increased the total production of spermidine, but also increased both the intracellular concentration of spermidine and the accumulation of RNA. Exogenous spermidine extensively relaxed RNA synthesis in amino acid-starved cultures of 15 TAU. Analysis in sucrose density gradients showed that the RNA accumulated in the presence of spermidine was ribosomal RNA.Cells of E. coli K-12 RC(rel) met(-) leu(-), grown in a complete medium, had approximately the same ratio of free spermidine to RNA as did strain 15 TAU. However, the relaxed strain showed a much lower ratio of putrescine to spermidine than the stringent 15 TAU. Omission of methionine stopped spermidine synthesis and markedly increased both the intracellular accumulation and the total production of putrescine. It seems that a high intracellular level of spermidine acts as a feedback inhibitor in the biosynthesis of putrescine in this strain. The hypothesis that the intracellular concentration of polyamines may participate in the control of the synthesis of ribosomal RNA in bacteria is discussed.     

The interaction between streptomycin and ribosomal RNA

The present study shows that a mutation in the 530 loop of 16S rRNA impairs the binding of streptomycin to the bacterial ribosome, thereby restricting the misreading effect of the drug. Previous reports demonstrated that proteins S4, S5 and S12 as well as the 915 region of 16S rRNA are involved in the binding of streptomycin, and indicated that the drug not only interacts with the 30S subunit but also with the 50S subunit. The relationship between the target of streptomycin and its known interference with the proofreading control of translational accuracy is examined in light of these results.