James J. Schmidt

Bio: James J. Schmidt is an academic researcher from United States Army Medical Research Institute of Infectious Diseases. The author has contributed to research in topics: Clostridium botulinum & Peptide. The author has an hindex of 32, co-authored 59 publications receiving 2562 citations. Previous affiliations of James J. Schmidt include United States Department of the Army.

Endoproteinase Activity of Type A Botulinum Neurotoxin: Substrate Requirements and Activation by Serum Albumin

Abstract: Type A botulinum neurotoxin, a zinc-dependent endoproteinase that selectively cleaves the neuronal protein SNAP-25, can also cleave relatively short peptides. We found that bovine and other serum albumins stimulated the type A-catalyzed hydrolysis of synthetic peptide substrates, through a direct effect on the kinetic constants of the reaction. Furthermore, with bovine serum albumin in the assays, the optimum substrate size was 16 residues (11 on the amino-terminal side of the cleavage site and 5 on the carboxy-terminal side). To further investigate the catalytic requirements of the neurotoxin, peptides were synthesized with various amino acid substitutions at the P5 through P5′ substrate sites. Changes at all of these locations affected values for both kcat and Km. Substitutions at the P2, P1′, and P2′ sites had more pronounced effects on hydrolysis rates than did substitutions at the P1 site. Enzyme–substrate interactions at the P3′ threonine probably involved the side-chain methyl group rather than the hydroxyl group. Replacing the P2′ alanine with leucine eliminated detectable hydrolysis, but not binding, since this peptide was an inhibitor. A negatively charged residue was preferred at P5, but not at P4. The data indicate that type A botulinum neurotoxin has an extended substrate recognition region and a requirement for arginine as the P1′ residue.

Identification of small molecule inhibitors of anthrax lethal factor

Abstract: The virulent spore-forming bacterium Bacillus anthracis secretes anthrax toxin composed of protective antigen (PA), lethal factor (LF) and edema factor (EF). LF is a Zn-dependent metalloprotease that inactivates key signaling molecules, such as mitogen-activated protein kinase kinases (MAPKK), to ultimately cause cell death. We report here the identification of small molecule (nonpeptidic) inhibitors of LF. Using a two-stage screening assay, we determined the LF inhibitory properties of 19 compounds. Here, we describe six inhibitors on the basis of a pharmacophoric relationship determined using X-ray crystallographic data, molecular docking studies and three-dimensional (3D) database mining from the US National Cancer Institute (NCI) chemical repository. Three of these compounds have Ki values in the 0.5–5 μM range and show competitive inhibition. These molecular scaffolds may be used to develop therapeutically viable inhibitors of LF.

Structure of the receptor binding fragment HC of tetanus neurotoxin.

Abstract: The 2.7 A structure of the tetanus neurotoxin receptor binding fragment Hc reveals a jelly-roll domain and a beta-trefoil domain. Hc retains the unique transport properties of the holotoxin and is capable of eliciting a protective immunological response against the full length holotoxin.

Proteolysis of synthetic peptides by type a botulinum neurotoxin

Abstract: Type A botulinum neurotoxin catalyzed the hydrolysis of synthetic peptides based on the sequence of the 25-kD synaptosomal protein SNAP-25. In each peptide, the toxin cleaved at a single glutaminyl-arginine bond corresponding to residues 197 and 198 of SNAP-25, confirming earlier reports on the enzymatic specificity of the toxin in synaptosomal preparations. Metal chelators inhibited catalysis, consistent with a metalloprotease activity. In contrast to tetanus toxin and other botulinum toxin serotypes, type A toxin hydrolyzed relatively short, 17-to 20-residue peptides. In the substrates, SNAP-25 residue 202 and one or more of residues 187–191 were required for efficient hydrolysis, but residues 167–186 and 203–206 were not. The highest rates of hydrolysis were found when the C-terminal residues of the peptides were amidated.

Fluorigenic Substrates for the Protease Activities of Botulinum Neurotoxins, Serotypes A, B, and F

Abstract: The seven botulinum neurotoxins (BoNTs) are zinc metalloproteases that cleave neuronal proteins involved in neurotransmitter release and are among the most toxic natural products known. High-throughput BoNT assays are needed for use in antibotulinum drug discovery and to characterize BoNT protease activities. Compared to other proteases, BoNTs exhibit unusually stringent substrate requirements with respect to amino acid sequences and polypeptide lengths. Nonetheless, we have devised a strategy for development of fluorigenic BoNT protease assays, based on earlier structure-function studies, that has proven successful for three of the seven serotypes: A, B, and F. In synthetic peptide substrates, the P 1 and P 3 ′ residues were substituted with 2,4-dinitrophenyl-lysine and S -( N -[4-methyl-7-dimethylamino-coumarin-3-yl]-carboxamidomethyl)-cysteine, respectively. By monitoring the BoNT-catalyzed increase in fluorescence over time, initial hydrolysis rates could be obtained in 1 to 2 min when BoNT concentrations were 60 ng/ml (about 1 nM) or higher. Each BoNT cleaved its fluorigenic substrate at the same location as in the neuronal target protein, and kinetic constants indicated that the substrates were selective and efficient. The fluorigenic assay for BoNT B was used to characterize a new competitive inhibitor of BoNT B protease activity with a K i value of 4 μM. In addition to real-time activity measurements, toxin concentration determinations, and kinetic studies, the BoNT substrates described herein may be directly incorporated into automated high-throughput assay systems to screen large numbers of compounds for potential antibotulinum drugs.

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Abstract: 抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

The NCI60 human tumour cell line anticancer drug screen

Abstract: The US National Cancer Institute (NCI) 60 human tumour cell line anticancer drug screen (NCI60) was developed in the late 1980s as an in vitro drug-discovery tool intended to supplant the use of transplantable animal tumours in anticancer drug screening. This screening model was rapidly recognized as a rich source of information about the mechanisms of growth inhibition and tumour-cell kill. Recently, its role has changed to that of a service screen supporting the cancer research community. Here I review the development, use and productivity of the screen, highlighting several outcomes that have contributed to advances in cancer chemotherapy.

Neurotoxins Affecting Neuroexocytosis

Abstract: Nerve terminals are specific sites of action of a very large number of toxins produced by many different organisms. The mechanism of action of three groups of presynaptic neurotoxins that interfere directly with the process of neurotransmitter release is reviewed, whereas presynaptic neurotoxins acting on ion channels are not dealt with here. These neurotoxins can be grouped in three large families: 1) the clostridial neurotoxins that act inside nerves and block neurotransmitter release via their metalloproteolytic activity directed specifically on SNARE proteins; 2) the snake presynaptic neurotoxins with phospholipase A(2) activity, whose site of action is still undefined and which induce the release of acethylcholine followed by impairment of synaptic functions; and 3) the excitatory latrotoxin-like neurotoxins that induce a massive release of neurotransmitter at peripheral and central synapses. Their modes of binding, sites of action, and biochemical activities are discussed in relation to the symptoms of the diseases they cause. The use of these toxins in cell biology and neuroscience is considered as well as the therapeutic utilization of the botulinum neurotoxins in human diseases characterized by hyperfunction of cholinergic terminals.

Anti-virulence strategies to combat bacteria-mediated disease

Abstract: Antibiotic resistance is one of the greatest challenges of the twenty-first century. However, the increasing understanding of bacterial pathogenesis and intercellular communication has revealed many potential strategies to develop novel drugs to treat bacteria-mediated disease. Interference with bacterial virulence and/or cell-to-cell signalling pathways is an especially compelling approach, as it is thought to apply less selective pressure for the development of bacterial resistance than traditional strategies, which are aimed at killing bacteria or preventing their growth. Here, we discuss the mechanisms of bacterial virulence and present promising anti-virulence strategies and compounds for the future treatment of bacterial infections.