Modeling the structure of mAb 14B7 bound to the anthrax protective antigen

The anthrax protective antigen (PA) is a key component of the tripartite anthrax toxin. Monoclonal antibody (mAb) 14B7 and its engineered, affinity-matured variants have been shown to be effective in blocking PA binding to cellular receptors and mitigating anthrax toxicity. Here, we perform computational structural modeling of the mAb 14B7-PA interaction. Our objectives are to determine the structure of the 14B7-PA complex, to deduce a structural explanation for the affinity maturation from the docking models, and to study the effect of inaccuracies in the antibody homology model on docking. We used the RosettaDock program to dock PA with the mAb 14B7 crystal structure or homology model. Our simulations generate two distinct binding orientations consistent with experimental residue mutations that diminish 14B7-PA binding. Furthermore, the models suggest new site-directed mutations to positively identify one of these two solutions as the correct 14B7-PA docking orientation. The models indicate that PA regions 648-660 and 712-720 may be important for 14B7 binding in addition to the known PA epitope, and the binding interfaces are similar to that seen in the PA complex with cellular receptor CMG2. Antibody residues involved in affinity maturation do not contact the antigen in the docking models, suggesting that affinity maturation in the 14B7 family does not result from direct enhancements of antibody-antigen contacts. Docking the homology model produces low-resolution representations of the crystal structure docking orientations, but homology model docking is frustrated by antibody H3 loop conformation errors. This work demonstrates the usefulness and limitations of computational structure prediction for the development of antibody therapeutics, and reemphasizes the need for flexible backbone docking algorithms to achieve high-resolution docking using homology models.     

肉毒毒素在肿瘤治疗和辅助治疗领域的研究进展

肉毒毒素是肉毒梭状芽胞杆菌生长繁殖过程中产生的一种细菌外毒素。它可以通过抑制相关神经递质的释放而抑制神经的生理作用,目前临床适应症涉及神经内科、整形外科、康复科、泌尿科等多领域。最新研究发现,肉毒毒素作为毒蕈碱受体拮抗剂可抑制迷走神经释放乙酰胆碱,从而抑制胃癌的发生与发展。同时发现肉毒毒素可通过阻断去甲肾上腺素等神经递质的释放引起神经性的血管舒张,从而打开肿瘤神经血管网来改善肿瘤的放射和化学治疗的疗效。现就其在肿瘤治疗及其辅助治疗相关领域的研究现状进行了综述。     

Function inferences from a molecular structural model of bacterial ParE toxin

Toxin-antitoxin (TA) systems contribute to plasmid stability by a mechanism that relies on the differential stabilities of the toxin and antitoxin proteins and leads to the killing of daughter bacteria that did not receive a plasmid copy at the cell division. ParE is the toxic component of a TA system that constitutes along with RelE an important class of bacterial toxin called RelE/ParE superfamily. For ParE toxin, no crystallographic structure is available so far and rare in vitro studies demonstrated that the target of toxin activity is E. coli DNA gyrase. Here, a 3D Model for E. coli ParE toxin by molecular homology modeling was built using MODELLER, a program for comparative modeling. The Model was energy minimized by CHARMM and validated using PROCHECK and VERIFY3D programs. Resulting Ramachandran plot analysis it was found that the portion residues failing into the most favored and allowed regions was 96.8%. Structural similarity search employing DALI server showed as the best matches RelE and YoeB families. The Model also showed similarities with other microbial ribonucleases but in a small score. A possible homologous deep cleft active site was identified in the Model using CASTp program. Additional studies to investigate the nuclease activity in members of ParE family as well as to confirm the inhibitory replication activity are needed. The predicted Model allows initial inferences about the unexplored 3D structure of the ParE toxin and may be further used in rational design of molecules for structure-function studies.     

Dopaminergic Inhibition of Prolactin Release and Calcium Influx Induced by Neurotensin in Anterior Pituitary Is Independent of Cyclic AMP System

The present study demonstrates that 3,4-dihydroxyphenylethylamine (DA, dopamine) prevents neurotensin (NT) stimulation of both prolactin (PRL) release and calcium influx by interacting with specific receptors that are functionally linked to calcium channels. As shown by the studies with dispersed cells from rat anterior pituitary, the pharmacology of the control of PRL release and calcium influx, both induced by NT, was found to be typical of a DAergic process. This was demonstrated by the order of potency of agonists in inhibiting PRL release and calcium influx (DA greater than epinephrine greater than norepinephrine much greater than isoproterenol); by the high affinity of antagonists such as haloperidol and fluphenazine for this process; and by the high degree of stereoselectivity of sulpiride. Specific D2 receptor agonists, such as bromocriptine and lisuride, and the specific D2 receptor antagonist (-)-sulpiride were found to be highly potent on the DA receptors negatively coupled with calcium channels and PRL release. DA was found to lack the capacity to change the influx of calcium induced by either the sodium channel activator veratridine or high extracellular potassium levels, thus indicating a specific action of this amine on calcium channels sensitive to NT. In a range of concentrations that are effective in inhibiting either the calcium influx or the PRL release, both induced by NT, DA did not alter the cyclic AMP generating system. DA (from 1.0 nM to 50 nM) did not affect adenylate cyclase activity in rat pituitary gland homogenates and did not modify intracellular cyclic AMP levels in pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Common antigenic determinant in the receptor binding sites of Escherichia coli enterotoxin and cholera toxin

Abstract A mutant (TUH No. 9) of a porcine strain of enterotoxigenic Escherichia coli (ETEC) produces as abnormal B subunit (B') of heat-labile enterotoxin (LT), which has aspartate instead of glycine at residue 33 from the N-terminus and does not bind to the receptor, GM1 ganglioside. The antigenicities of the receptor-binding site of LT were analyzed.
The antibody, which could not bind to the B' subunit in the anti-B subunit of porcine LT(LTp)-serum, could bind to cholera toxin (CT), LTp and LT produced by a human ETEC strain (LTh), suggesting that it recognizes a common epitope of LTp, LTh and CT. Thus glycine at residue 33 from the N-terminus in the B subunit of CT, LTh and LTp may be related to the common epitope of these three toxins. The bindings of CT, LTh and LTp to the antibody were inhibited by the GM1 ganglioside.
These data indicate that the antibody recognizes a common epitope in the receptor (GM1 ganglioside)-binding site of CT, LTh and LTp.     

Adsorption of insecticidal toxin from Bacillus thuringiensis subsp. Kurstaki by some Chinese soils: effects of organic acid ligands addition

We have investigated the effects of low molecular weight organic acid ligands on the adsorption of the insecticidal toxin from Bacillus thuringiensis (Bt) by the colloidal (<2 μm particle-size) fraction of some soils. The desorption of the bound toxin by NaCl and phosphate buffer has also been measured. The soils used were a red soil (Ultisol), a latosol (Oxisol), a yellow brown soil (Alfisol) and a yellow cinnamon soil (Alfisol) from central and southern China. The adsorption isotherms were all of the L-type, and the data fitted the Langmuir equation (R² > 0.97). When present at low concentrations, organic acids (acetate, oxalate, citrate) had an inhibitory effect on toxin adsorption. Uptake, however, was promoted when the organic acid concentration exceeded 10 mM. The toxin was very strongly bound by the soils but the soil-toxin interaction weakened in the presence of organic acids. A small portion of the toxin was adsorbed by electrostatic and ligand exchange interactions. The addition of organic acids appeared to enhance these interactions. Responsible Editor: Thomas B. Kinraide     

Polymerase chain reaction detection of Clostridium perfringens in feces from captive and wild chimpanzees, Pan troglodytes

Background For veterinary management of non‐human primates in captivity, and conservation of wild‐living primates, management of their health risks is necessary. Incidences of pathogenic bacteria in the fecal specimens are considered as one of the useful indicators for non‐invasive health monitoring. Methods We carried out the detection of Clostridium perfringens in feces from captive and wild chimpanzees by the rapid polymerase chain reaction method. Results The bacterium was detected in most fecal specimens (80%) in captive chimpanzees. Contrarily, the detection rate in the wild chimpanzees was low, with 23% (n = 12) of 53 fecal samples from the Bossou group, Guinea, and 1.2% (1/81) in the Mahale group, Tanzania. Conclusions These results show that the intestinal microflora differs between Pan populations under various living conditions, being influenced by their diet and environment.     

Pollen-specific expression of theArabidopsis thaliana α1-tubulin promoter assayed by β-glucuronidase,chloramphenicol acetyltransferase and diphtheria toxin reporter genespromoter assayed by β-glucuronidase,chloramphenicol acetyltransferase and diphtheria toxin reporter genes

We have characterized the promoter specificity of theArabidopsis thaliana α₁-tubulin (α ₁-tub) gene by studying expression patterns of gene fusions between the 2.2 kbp 5′ upstream region of theα ₁-tub gene and each of three different reporters: chloramphenical acetyltransferase, β-glucuronidase or the diphtheria toxin chain A gene. Analysis of transgenic tobacco andArabidopsis plants carrying the transgene showed that the chloramphenicol acetyltransferase and β-glucuronidase activities were not detected in any vegetative or reproductive organs except mature pollen. Transgenic tobacco plants carrying the diphtheria toxin chain A gene under the control of theα ₁-tub promoter were of normal phenotype but seed fertility was drastically reduced. Furthermore, the transgene could not be transmitted to the next generation through pollen, supporting the observation that theα ₁-tub promoter is active only in pollen. It was observed that the promoter activity was most active in mature pollen and decreased significantly duringin vitro pollen germination, indicating that the promoter is inactive or subdued in germinating pollen. The promoter activity was not affected by various plant growth hormones during pollen maturation.