Monoclonal antibody to shiga toxin 1, which blocks receptor binding and neutralizes cytotoxicity

A monoclonal antibody, 5-5B, which neutralizes Shiga toxin 1 (Stx1) cytotoxicity of Escherichia coli, was constructed. An epitope analysis indicated that Asn55 in Stx1 B subunit was an important residue. This result and our previous results using an anti-Stx2 monoclonal antibody indicate that the region around the cysteine residue of the disulfide bond might be important for the neutralization of Stx cytotoxicity, making it a potential vaccination candidate.     

Roles of Long-range Electrostatic Domain Interactions and K+ in Phosphoenzyme Transition of Ca2+-ATPase

Sarcoplasmic reticulum Ca²⁺-ATPase couples the motions and rearrangements of three cytoplasmic domains (A, P, and N) with Ca²⁺ transport. We explored the role of electrostatic force in the domain dynamics in a rate-limiting phosphoenzyme (EP) transition by a systematic approach combining electrostatic screening with salts, computer analysis of electric fields in crystal structures, and mutations. Low KCl concentration activated and increasing salt above 0.1 m inhibited the EP transition. A plot of the logarithm of the transition rate versus the square of the mean activity coefficient of the protein gave a linear relationship allowing division of the activation energy into an electrostatic component and a non-electrostatic component in which the screenable electrostatic forces are shielded by salt. Results show that the structural change in the transition is sterically restricted, but that strong electrostatic forces, when K⁺ is specifically bound at the P domain, come into play to accelerate the reaction. Electric field analysis revealed long-range electrostatic interactions between the N and P domains around their hinge. Mutations of the residues directly involved and other charged residues at the hinge disrupted in parallel the electric field and the structural transition. Favorable electrostatics evidently provides a low energy path for the critical N domain motion toward the P domain, overcoming steric restriction. The systematic approach employed here is, in general, a powerful tool for understanding the structural mechanisms of enzymes.     

Molecular identification of the causative agent of human strongyloidiasis acquired in Tanzania: Dispersal and diversity of Strongyloides spp. and their hosts

In order to identify the causative agent of imported strongyloidiasis found in a Japanese mammalogist, who participated in a field survey in Tanzania, the hyper-variable region IV (HVR-IV) of 18S ribosomal DNA and partial mitochondrial cytochrome c-oxidase subunit 1 gene (cox1) were analyzed and compared with Strongyloides fuelleborni collected from apes and monkeys of Africa and Japan, and S. stercoralis from humans, apes and dogs. The HVR-IV and cox1 of the patient's worms were identical to or only slightly differed from those of worms parasitic in Tanzanian chimpanzees and yellow baboons, demonstrating that the patient acquired the infection during her field survey in Tanzania. Phylogenetic analysis with the maximum-likelihood method largely divided isolates of S. fuelleborni into three groups, which corresponded to geographical localities but not to host species. Meanwhile, isolates of S. stercoralis were grouped by the phylogenetic analysis into dog-parasitic and primate-parasitic clades, and not to geographical regions. It is surmised that subspeciation has occurred in S. fuelleborni during the dispersal of primates in Africa and Asia, while worldwide dispersal of S. stercoralis seems to have occurred more recently by migration and the activities of modern humans.     

S-(2-(acylamino)phenyl) 2,2-dimethylpropanethioates as CETP inhibitors

Studies on the relationship between the structure of the benzene moiety of S-(2-(acylamino)phenyl) 2,2-dimethylpropanethioates and CETP inhibitory activity were performed. Substituents on the benzene moiety influenced CETP inhibitory activity in a type and position dependent manner, and electron-withdrawing groups at the 4- or 5-position increased the activity. The most potent compound showed 50% inhibition of CETP activity in human plasma at a concentration of 2 microM.     

Properties of the PriA helicase domain and its role in binding PriA to specific DNA structures

Primosome assembly protein PriA functions in the assembly of the replisome at forked DNA structures. Whereas its N-terminal DNA binding domain (DBD) binds independently to DNA, the affinity of DBD protein for forked structures is relatively weak. Although the PriA helicase domain (HD) is required for high affinity fork binding, HD protein had very low affinity for DNA. It had only low levels of ATPase activity, and it hydrolyzed ATP when DNA was absent whereas PriA did not. HD catalyzed unwinding of a minimal substrate composed of a duplex with a 3' single-stranded tail. Single-strand binding protein (SSB) bound to the tail of this substrate inhibited this reaction by full-length PriA but enhanced the reaction by HD. SSB stabilized binding of PriA but not of DBD or HD to duplexes with a 5' or 3' single-stranded tail. On forked substrates SSB enhanced helicase action on the lagging-strand arm by PriA but not by HD. The results indicate that synergy of the DBD and HD allows stable binding at the interface between duplex and single-stranded DNA bound by SSB. This mode of binding may be analogous to fork binding, which orients the helicase to act on the lagging-strand side of the fork.     

The endosome-lysosome system in the absorptive cells of goldfish hindgut

Summary The vacuolar system in the absorptive cells of the goldfish hindgut was studied by rapid freeze-substituted and cytochemical techniques. The apical cytoplasm of the absorptive cells contained two types of vacuoles: endosomes and lysosomes. The former were characterized by an absence of acid phosphatase activity, a dot-like distribution of material at the peripheral rim, the labelling of the inner surface with horseradish peroxidase (HRP), and by frequent connections to cytoplasmic tubules (CT), which were also free of acid phosphatase activity. The latter vacuole was preferentially located in the deeper cytoplasm and was characterized by the presence of acid phosphatase activity, an electron-dense interior matrix, a peripheral electron-lucent region (a halo), and by the detachment of HRP from the inner surface. Connections between CTs and these latter vacuoles were rarely seen. In the deeper cytoplasm, fusion between endosomes and lysosomes was sometimes observed. These results suggest that the vacuoles which are associated with CTs are endosomes, but not lysosomes, and that internalized materials are transported through the endosome-lysosome system to a giant food vacuole in the cell.