Identification of residues linked to the slow-->fast transition of thrombin.

Residues energetically linked to the allosteric transition of thrombin from its anticoagulant slow form to the procoagulant fast form have been identified by site-directed mutagenesis. The energetics of recognition by the two forms of the enzyme were probed by using a synthetic chromogenic substrate, fibrinogen, and hirudin. The thrombin residues E39, W60d, E192, D221, and D222 are linked to the slow-->fast transition and are part of an "allosteric core" through which events originating at the Na+ binding loop propagate to other regions of the enzyme. The thrombin residues Y76, W96, W148, and R173 lie at the periphery of the allosteric core, affect recognition of fibrinogen and hirudin to the same extent in both forms, and are not linked to the slow-->fast transition.

Rapid and efficient fusion of phospholipid vesicles by the α-helical core of a SNARE complex in the absence of an N-terminal regulatory domain

A protease-resistant core domain of the neuronal SNARE complex consists of an α-helical bundle similar to the proposed fusogenic core of viral fusion proteins [Skehel, J. J. & Wiley, D. C. (1998) Cell 95, 871–874]. We find that the isolated core of a SNARE complex efficiently fuses artificial bilayers and does so faster than full length SNAREs. Unexpectedly, a dramatic increase in speed results from removal of the N-terminal domain of the t-SNARE syntaxin, which does not affect the rate of assembly of v-t SNARES. In the absence of this negative regulatory domain, the half-time for fusion of an entire population of lipid vesicles by isolated SNARE cores (≈10 min) is compatible with the kinetics of fusion in many cell types.

Large-scale isolation of candidate virulence genes of Pseudomonas aeruginosa by in vivo selection.

Pseudomonas aeruginosa, an opportunistic human pathogen, is a major causative agent of mortality and morbidity in immunocompromised patients and those with cystic fibrosis genetic disease. To identify new virulence genes of P. aeruginosa, a selection system was developed based on the in vivo expression technology (IVET) that was first reported in Salmonella system. An adenine-requiring auxotrophic mutant strain of P. aeruginosa was isolated and found avirulent on neutropenic mice. A DNA fragment that can complement the mutant strain, containing purEK operon that is required for de novo biosynthesis of purine, was sequenced and used in the IVET vector construction. By applying the IVET selection system to a neutropenic mouse infection model, genetic loci that are specifically induced in vivo were identified. Twenty-two such loci were partially sequenced and analyzed. One of them was a well-studied virulence factor, pyochelin receptor (FptA), that is involved in iron acquisition. Fifteen showed significant homology to reported sequences in GenBank, while the remaining six did not. One locus, designated np20, encodes an open reading frame that shares amino acid sequence homology to transcriptional regulators, especially to the ferric uptake regulator (Fur) proteins of other bacteria. An insertional np20 null mutant strain of P. aeruginosa did not show a growth defect on laboratory media; however, its virulence on neutropenic mice was significantly reduced compared with that of a wild-type parent strain, demonstrating the importance of the np20 locus in the bacterial virulence. The successful isolation of genetic loci that affect bacterial virulence demonstrates the utility of the IVET system in identification of new virulence genes of P. aeruginosa.

Subparsec-scale structure and evolution of Centaurus A (NGC5128).

We present a series of 8.4-GHz very-long-baseline radio interferometry images of the nucleus of Centaurus A (NGC5128) made with a Southern Hemisphere array, representing a 3.3-year monitoring effort. The nuclear radio jet is approximately 50 milliarcseconds in extent, or at the 3.5-megaparsec distance of NGC5128, approximately 1 parsec in length. Subluminal motion is seen and structural changes are observed on time scales shorter than 4 months. High-resolution observations at 4.8 and 8.4 GHz made in November 1992 reveal a complex morphology and allow us to unambiguously identify the self-absorbed core located at the southwestern end of the jet.