Holomycin, a member of the pyrrothine class of antibiotics, displayed broad-spectrum antibacterial activity, inhibiting a variety of gram-positive and gram-negative bacteria, with the exception of Enterobacter cloacae, Morganella morganii, and Pseudomonas aeruginosa. The antibiotic lacked activity against the eukaryotic microorganisms Saccharomyces cerevisiae and Candida kefyr. Holomycin exhibited a bacteriostatic response against Escherichia coli that was associated with rapid inhibition of RNA synthesis in whole cells. Inhibition of RNA synthesis could have been a secondary consequence of inhibiting tRNA aminoacylation, thereby inducing the stringent response. However, the levels of inhibition of RNA synthesis by holomycin were similar in a stringent and relaxed pair of E. coli strains that were isogenic except for the deletion of the relA gene. This suggests that inhibition of RNA synthesis by holomycin could reflect direct inhibition of DNA-dependent RNA polymerase. Examination of the effects of holomycin on the kinetics of the appearance of ␤-galactosidase in induced E. coli cells was also consistent with inhibition of RNA polymerase at the level of RNA chain elongation. However, holomycin only weakly inhibited E. coli RNA polymerase in assays using synthetic poly(dA-dT) and plasmid templates. Furthermore, inhibition of RNA polymerase was observed only at holomycin concentrations in excess of those required to inhibit the growth of E. coli. It is possible that holomycin is a prodrug, requiring conversion in the cell to an active species that inhibits RNA polymerase.Thiolutin and holomycin (see Fig. 1) are members of the pyrrothine class of naturally occurring antibiotics that are characterized by the possession of a unique pyrrolinonodithiole nucleus (2). Although these antimicrobial agents were originally discovered more than 40 years ago (6, 13, 23, 26), relatively little is known about their mode of action. Limited studies with thiolutin suggest that the pyrrothines may act as inhibitors of DNA-dependent RNA polymerase. Thus, thiolutin preferentially inhibits RNA synthesis in both Saccharomyces cerevisiae (10) and Escherichia coli (14) and is reported to be a potent inhibitor of partially purified RNA polymerases from S. cerevisiae (10, 25). Furthermore, by monitoring the effects of thiolutin on the induction of ␤-galactosidase in E. coli, Khachatourians and Tipper (14) concluded that the antibiotic interfered with RNA chain elongation rather than with initiation of transcription.However, there are several observations that cast doubt upon the hypothesis that thiolutin, and therefore the pyrrothines as a class, prevents microbial growth by interfering with the elongation of RNA transcripts catalyzed by RNA polymerase. For instance, Sivasubramanian and Jayaraman (24) were unable to reproduce the observations made by Khachatourians and Tipper (14) concerning the effects of thiolutin on ␤-galactosidase induction in E. coli. Thus, these authors (24) concluded that thiolutin inhibits initiation of RNA tran...

Nuclear magnetic resonance (NMR) spectroscopy was used to investigate the transfer of sialic acid from a range of sialic acid donor compounds to acceptor molecules, catalyzed by Trypanosoma cruzi trans-sialidase (TcTS). We demonstrate here that NMR spectroscopy is a powerful tool to monitor the trans-sialidase enzyme reaction for a variety of donor and acceptor molecules. The hydrolysis or transfer reactions that are catalyzed by TcTS were also investigated using a range of N-acetylneuraminosyl-based donor substrates and asialo acceptor molecules. These studies showed that the synthetic N-acetylneuraminosyl donor 4-methylumbelliferyl alpha-d-N-acetylneuraminide (MUN) is hydrolyzed by the enzyme approximately 3-5 times faster than either the disaccharide Neu5Acalpha(2,3)Galbeta1Me or the trisaccharide Neu5Acalpha(2,3)Lacbeta1Me. In the transfer reaction, we show that Neu5Acalpha(2,3)Lacbeta1Me is the most favorable substrate for TcTS and is a better substrate than the naturally-occurring N-acetylneuraminosyl donor alpha1-acid glycoprotein. In the case of MUN as the donor molecule, the transfer of Neu5Ac to different acceptors is significantly slower than when other N-acetylneuraminosyl donors are used. We hypothesize that when MUN is bound by the enzyme, the orientation and steric bulk of the umbelliferyl aglycon moiety may restrict the access for the correct positioning of an acceptor molecule. AutoDock studies support our hypothesis and show that the umbelliferyl aglycon moiety undergoes a strong pi-stacking interaction with Trp-312. The binding properties of TcTS towards acceptor (lactose) and donor substrate (Neu5Ac) molecules have also been investigated using saturation transfer difference (STD) NMR experiments. These experiments, taken together with other published data, have clearly demonstrated that lactose in the absence of other coligands does not bind to the TcTS active site or other binding domains. However, in the presence of the sialic acid donor, lactose (an asialo acceptor) was observed by NMR spectroscopy to interact with the enzyme's active site. The association of the asialo acceptor with the active site is an absolute requirement for the transfer reaction to proceed.

This paper presents the results of an action research study into the acute care experience of Dissociative Identity Disorder. The study, which was grounded in principles of critical social science, utilized focus group interviews and narrative construction. Nurses and patients are under-represented in all clinical evaluation and their voices need to be heard if services are to be truly collaborative. Findings of the study extend intrapsychic theories of trauma to emphasize the interpersonal relationship between nurse and person who can work together to facilitate recovery from trauma, make connections both intra and interpersonally and build resilience.

The biological properties of SB 205952, a nitrofuryl oxazole derivative of monic acid, differ from those of the closely related antibacterial agent mupirocin. Compared with mupirocin, SB 205952 has increased antimicrobial potency, an extended spectrum including mupirocin-resistant staphylococci, and rapid bactericidal activity. SB 205952, like mupirocin, is a potent inhibitor of bacterial isoleucyl-tRNA synthetase (IRS) in mupirocinsusceptible organisms but does not inhibit IRS from mupirocin-resistant staphylococci, indicating that SB 205952 has more than one mechanism of action. SB 205952 rapidly inhibits protein, RNA, and DNA syntheses in mupirocin-susceptible and mupirocin-resistant staphylococci. In each case, the effect on RNA synthesis is relaxed by treatment with chloramphenicol, indicating that inhibition of RNA synthesis is probably a secondary consequence of stringent control. It is proposed that SB 205952 possesses one or more mechanisms of action in addition to IRS inhibition, probably mediated by its nitrofuryl component.

Saturation transfer difference (STD) (1)H NMR experiments were used to probe the epitope binding characteristics of the sialidase [EC 3.2.1.18] from the bacterium Vibrio cholerae, the causative agent of cholera. Binding preferences were investigated for N-acetylneuraminic acid (Neu5Ac, 1), the product of the sialidase catalytic reaction, for the known sialidase inhibitor 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enoic acid (Neu5Ac2en, 2), and for the uronic acid-based Neu5Ac2en mimetic iso-propyl 2-acetamido-2,4-dideoxy-alpha-L-threo-hex-4-enopyranosiduronic acid (3), in which the native glycerol side-chain of Neu5Ac2en is replaced with an O-iso-propyl ether. The STD experiments provided evidence, supporting previous studies, that Neu5Ac (1) binds to the sialidase as the alpha-anomer. Docking experiments using DOCK (version 4.0.1) revealed further information regarding the binding characteristics of the enzyme active site in complex with Neu5Ac2en (2) and the Neu5Ac2en mimetic (3), indicating an expected dominant interaction of the acetamide moiety with the protein.