Molecular Cloning of a Bovine Immunoglobulin Lambda Chain cDNA

A cDNA library of the bovine mammary gland constructed in pBR322 was screened by mRNA hybrid-selected translation and by differential hybridization. Several immunoglobulin (Ig) λ light-chain clones were identified and sequenced. Nucleotide sequence comparison of bovine and human Ig λ chains showed a high degree of homology for constant regions and for J regions. The amino acid (aa) sequence encoded by the constant region of the bovine Ig λ chain cDNA contains 107 aa with differences at 24 aa positions from the human Ig A chain. Three complementarity-determining regions (CDR1,2,3) characteristic of the variable region of bovine Ig λ chain cDNA can be distinguished. The bovine and human sequences display good homology in the framework region 3 (FR3) but only patches of homology throughout the FR2 region. The 5′ end of the bovine Ig λ chain cDNA fragment of clone 1-14E contains five stop codons: two in CDR1, one in FR1 and two in the hydrophobic prepeptide region. These data suggest that the Igλ mRNA of clone 1-14E is transcribed from the Vλ pseudogene

Tailored ß-Cyclodextrin Blocks the Translocation Pores of Binary Exotoxins from C. Botulinum and C. Perfringens and Protects Cells from Intoxication

International audienceBackgroundClostridium botulinum C2 toxin and Clostridium perfringens iota toxin are binary exotoxins, which ADP-ribosylate actin in the cytosol of mammalian cells and thereby destroy the cytoskeleton. C2 and iota toxin consists of two individual proteins, an enzymatic active (A-) component and a separate receptor binding and translocation (B-) component. The latter forms a complex with the A-component on the surface of target cells and after receptor-mediated endocytosis, it mediates the translocation of the A-component from acidified endosomal vesicles into the cytosol. To this end, the B-components form heptameric pores in endosomal membranes, which serve as translocation channels for the A-components.Here we demonstrate that a 7-fold symmetrical positively charged ß-cyclodextrin derivative, per-6-S-(3-aminomethyl)benzylthio-ß-cyclodextrin, protects cultured cells from intoxication with C2 and iota toxins in a concentration-dependent manner starting at low micromolar concentrations. We discovered that the compound inhibited the pH-dependent membrane translocation of the A-components of both toxins in intact cells. Consistently, the compound strongly blocked transmembrane channels formed by the B-components of C2 and iota toxin in planar lipid bilayers in vitro. With C2 toxin, we consecutively ruled out all other possible inhibitory mechanisms showing that the compound did not interfere with the binding of the toxin to the cells or with the enzyme activity of the A-component.Conclusions/SignificanceThe described ß-cyclodextrin derivative was previously identified as one of the most potent inhibitors of the binary lethal toxin of Bacillus anthracis both in vitro and in vivo, implying that it might represent a broad-spectrum inhibitor of binary pore-forming exotoxins from pathogenic bacteria

In Vivo Efficacy of β-Cyclodextrin Derivatives against Anthrax Lethal Toxin▿

We evaluated the in vivo efficacy of three β-cyclodextrin derivatives that block the anthrax protective antigen pore. These compounds were at least 15-fold more potent than previously described β-cyclodextrins in protecting against anthrax lethal toxin in a rat model. One of the drugs was shown to protect mice from bacterial infection

Symmetry Requirements for Effective Blocking of Pore-Forming Toxins: Comparative Study with α-, β-, and γ-Cyclodextrin Derivatives ▿ †

We compared the abilities of structurally related cationic cyclodextrins to inhibit Bacillus anthracis lethal toxin and Staphylococcus aureus α-hemolysin. We found that both β- and γ-cyclodextrin derivatives effectively inhibited anthrax toxin action by blocking the transmembrane oligomeric pores formed by the protective antigen (PA) subunit of the toxin, whereas α-cyclodextrins were ineffective. In contrast, α-hemolysin was selectively blocked only by β-cyclodextrin derivatives, demonstrating that both symmetry and size of the inhibitor and the pore are important

Inhibition of Anthrax Protective Antigen Outside and Inside the Cell

In the course of Bacillus anthracis infection, B. anthracis lethal factor (LF) and edema factor bind to a protective antigen (PA) associated with cellular receptors ANTXR1 (TEM8) or ANTXR2 (CMG2), followed by internalization of the complex via receptor-mediated endocytosis. A new group of potential antianthrax drugs, β-cyclodextrins, has recently been described. A member of this group, per-6-(3-aminopropylthio)-β-cyclodextrin (AmPrβCD), was shown to inhibit the toxicity of LF in vitro and in vivo. In order to determine which steps in lethal factor trafficking are inhibited by AmPrβCD, we developed two targeted fluorescent tracers based on LFn, a catalytically inactive fragment of LF: (i) LFn site specifically labeled with the fluorescent dye AlexaFluor-594 (LFn-Al), and (ii) LFn-decorated liposomes loaded with the fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (LFn-Lip). Both tracers retained high affinity to PA/ANTXR complexes and were readily internalized via receptor-mediated endocytosis. Using fluorescent microscopy, we found that AmPrβCD inhibits receptor-mediated cell uptake but not the binding of LFn-Al to PA/ANTXR complexes, suggesting that AmPrβCD works outside the cell. Moreover, AmPrβCD and LFn-Al synergistically protect RAW 264.7 cells from PA-mediated LF toxicity, confirming that AmPrβCD did not affect the binding of LFn-Al to receptor-associated PA. In contrast, AmPrβCD did not inhibit PA-mediated internalization of LFn-Lip, suggesting that multiplexing of LFn on the liposomal surface overcomes the inhibiting effects of AmPrβCD. Notably, internalized LFn-Al and LFn-Lip protected cells that overexpressed anthrax receptor TEM8 from PA-induced, LF-independent toxicity, suggesting an independent mechanism for PA inhibition inside the cell. These data suggest the potential for the use of β-cyclodextrins in combination with LFn-Lip loaded with antianthrax drugs against intracellular targets

Cationic quaternized aminocalix[4]arenes: Cytotoxicity, haemolytic and antibacterial activities

This study reports the characterization of three cationic amphiphillic aminocalix[4]arenes as potential antimicrobial agents in vitro. In cytotoxicity tests on mouse macrophage RAW 264.7 cells aminocalix[4]arenes 1 and 3 showed no toxicity up to 200 and 100 μM concentrations, respectively, while 2 was non-toxic only up to 50 μM. With regard to the haemolytic activity on rabbit red blood cells, 1 was not active at concentrations up to 100 μM in contrast to the other two studied macrocycles. Compounds showed negligible ability to protect either mouse macrophage RAW 264.7 cells from anthrax lethal toxin of Bacillus anthracis (B. anthracis) or rabbit red blood cells from α-haemolysin of Staphylococcus aureus (S. aureus) in comparison to amino-β-cyclodextrins. However, all aminocalix[4]arenes showed potential as antimicrobials. Their minimum inhibitory concentrations (MIC) against Escherichia coli (E. coli) and S. aureus were in the 16-32 μg/ml concentration range, while minimum lethal concentrations (MLC) varied from 16 to 256 μg/ml depending on the bacteria and aminocalix[4]arene considered. Macrocycle 1 showed partial synergism against S. aureus in tandem with a model antibacterial drug, fusidic acid, at certain concentration combinations