Aerolysin is a channel-forming toxin that must oligomerize in order to become insertion-competent. Modeling based on the crystal structure of the proaerolysin dimer and electron microscopic images of the oligomer indicated that a loop in domain 3 must move away from the beta-sheet that forms the main body of the protein before oligomerization can proceed. In order to determine if movement actually occurs, strategically located amino acids in the loop and in the sheet were replaced with cysteines by site-directed mutagenesis. A double mutant was produced in which the new cysteines, at position 253 on the loop and position 300 in the sheet, were close enough together to allow formation of a disulfide bridge. The double mutant was unable to oligomerize, and it was completely inactive, showing not only that the bridge had formed but also that movement of the loop was essential for formation of the oligomer. The existence of the bridge was confirmed by X-ray crystallography. The reduced form of the protein and the single mutants T253C and A300C were as active as wild type, indicating that the amino acid replacements themselves had no functional consequences. Labeling studies using an environment-sensitive fluorescent sulfhydryl-reactive probe confirmed that the structure of the protein changes in the loop region as a consequence of proteolytic activation of proaerolysin, a step which also must precede oligomerization.

No abstract

Two hundred and eleven dogs (including strictly house and stray dogs) and 170 cattle in and around the city of Madras, India were screened for the presence of dermatophytosis. 106 strains of dermatophytes (89 strains from dogs and 17 strains from bovines) were isolated. 57/106 strains were Trichophyton mentagrophytes var. mentagrophytes and 42/106 strains were of the Microsporum gypseum complex. 5 strains of T. rubrum and 2 strains of T. simii were also obtained in culture. A predominance of M. gypseum complex isolates was recorded in stray dogs and cattle and T. mentagrophytes var. mentagrophytes and T. rubrum in strictly house dogs. The family history of the owners of the most of the dogs had clear records of dermatophytosis. Further, the owners of the 11 dogs that yielded T. mentagrophytes var. mentagrophytes had either tinea corporis or tinea pedis. The etiological agent of all the 11 human cases was T. mentagrophytes var. interdigitale. Similarly the owners of 4 of the 5 dogs that yielded T. rubrum were known T. rubrum patients. All these patients responded to oral griseofulvin or ketaconozole, but the recurrence of lesions was noted with the cessation of treatment. None of the patients had onychomycosis and the family history of all the patients revealed no reports of T. rubrum infections. The pet dogs were presumed to be the source of re-infection. Reversed transmission of dermatophytes from humans to animals may be the reason for the selective predominance of these organisms in strictly house dogs. They also may act as sources of reinfection. Most of the animals had small, occult, scattered lesions. These lesions may either go unnoticed or are ignored by the owners of the animals. The taxonomic status of T. mentagrophytes and T. mentagrophytes var. interdigitale was aligned to their teleomorph Arthroderma vanbreuseghemii. Our study suggests that the periodic screening and medication of all live-stock are essential for the prevention and management of the public health problem caused by dermatophytes.

The NMR structure of 2¢,5¢ d(GGGGCCCC) was determined to gain insights into the structural differences between 2¢,5¢-and 3¢,5¢-linked DNA duplexes that may be relevant in elucidating nature's choice of sugar-phosphate links to encode genetic information. The oligomer assumes a duplex with extended nucleotide repeats formed out of mostly N-type sugar puckers. With the exception of the 5¢-terminal guanine that assumes the syn glycosyl conformation, all other bases prefer the anti glycosyl conformation. Base pairs in the duplex exhibit slide ()1.96 Å ) and intermediate values for X-displacement ()3.23 Å ), as in ADNA, while their inclination to the helical axis is not prominent. Major and minor grooves display features intermediate to A and BDNA. The duplex structure of iso d(GGGGCCCC) may therefore be best characterized as a hybrid of A and BDNA. Importantly, the results confirm that even 3¢ deoxy 2¢,5¢ DNA supports duplex formation only in the presence of distinct slide ( ‡ )1.6 Å ) and X-displacement ( ‡ )2.5 Å ) for base pairs, and hence does not favor an ideal BDNA topology characterized by their near-zero values. Such restrictions on base pair movements in 2¢,5¢ DNA, which are clearly absent in 3¢,5¢ DNA, are expected to impose constraints on its ability for deformability of the kind observed in DNA during its compaction and interaction with proteins. It is therefore conceivable that selection pressure relating to the optimization of topological features might have been a factor in the rejection of 2¢,5¢ links in preference to 3¢,5¢ links.Keywords: structure of 2¢,5¢ DNA; evolution of 3¢,5¢ vs. 2¢,5¢ links in nucleic acids; AB hybrid structure; restrained base pair movements; topological restrictions in 2¢,5¢ DNA.Nature's selection of 3¢,5¢ linkages (instead of 2¢,5¢ linkages) in nucleic acids, to encode genetic information, is intriguing. The fact that 2¢,5¢ links are formed in abundance and serve as a template in nonenzymatic reactions suggest that they might have been the ancestors of the biotic 3¢,5¢ links, which could have evolved from a pool of 3¢,5¢ and 2¢,5¢ links [1]. Nucleic acids with 2¢,5¢ links satisfy one of the critical features required for the fidelity of replication, namely that they associate to form Watson and Crick base-paired duplex structures [2][3][4][5], although with weaker affinity than 3¢,5¢-linked DNA strands. However, detailed knowledge about stereochemistry, polymorphism and topological properties of 2¢,5¢ DNA duplexes, which may provide insights into the factors that determine nature's choice of sugar-phosphate links from a stereochemical perspective, is sparse [6][7][8][9]. In fact, there are only two reports of NMR structure determination -one on a 2¢,5¢ DNA fragment [10] and one on a 2¢,5¢ RNA fragment [11] -both of which suggest an A-type duplex structure with some stereochemical details that differ from genomic DNA and RNA duplexes. In this context, it is relevant to recognize the results from recent modeling studies on 2¢,5¢ nucleic acids, which suggest that 2¢,5¢ DNA can...