Mutations in the gene for aspartoacylase (ASPA), which catalyzes deacetylation of N-acetyl-L-aspartate in the central nervous system (CNS), result in Canavan Disease, a fatal dysmyelinating disease. Consistent with its role in supplying acetate for myelin lipid synthesis, ASPA is thought to be cytoplasmic. Here we describe the occurrence of ASPA within nuclei of rat brain and kidney, and in cultured rodent oligodendrocytes. Immunohistochemistry showed cytoplasmic and nuclear ASPA staining, the specificity of which was demonstrated by its absence from tissues of the Tremor rat, an ASPA-null mutant. Subcellular fractionation analysis revealed low enzyme activity against NAA in nuclear fractions from normal rats. Whereas two recent reports have indicated that ASPA exists as a dimer, size-exclusion chromatography of subcellular fractions showed ASPA is an active monomer in both subcellular fractions. Western blotting detected ASPA as a single 38 kD band. Because ASPA is small enough to passively diffuse into the nucleus, we constructed, expressed, and detected in COS-7 cells a green fluorescent protein-human ASPA (GFP-hASPA) fusion protein larger than the permissible size for the nuclear pore complex. GFP-hASPA was enzymatically active and showed mixed nuclear-cytoplasmic distribution. We conclude that ASPA is a regulated nuclear-cytoplasmic protein that may have distinct functional roles in the two cellular compartments.

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Plantazolicin (PZN) is a ribosomally synthesized and post-translationally modified natural product from Bacillus methylotrophicus FZB42 and Bacillus pumilus. Extensive tailoring to twelve of the fourteen amino acid residues in the mature natural product endows PZN with not only a rigid, polyheterocyclic structure, but also antibacterial activity. Here we report a remarkably discriminatory activity of PZN toward Bacillus anthracis, which rivals a previously-described gamma (γ) phage lysis assay in distinguishing B. anthracis from other members of the Bacillus cereus group. We evaluate the underlying cause of this selective activity by measuring the RNA expression profile of PZN-treated B. anthracis, which revealed significant upregulation of genes within the cell envelope stress response. PZN depolarizes the B. anthracis membrane like other cell envelope-acting compounds but uniquely localizes to distinct foci within the envelope. Selection and whole-genome sequencing of PZN-resistant mutants of B. anthracis implicate a relationship between the action of PZN and cardiolipin (CL) within the membrane. Exogenous CL increases the potency of PZN in wild type B. anthracis and promotes the incorporation of fluorescently tagged PZN in the cell envelope. We propose that PZN localizes to and exacerbates structurally compromised regions of the bacterial membrane, which ultimately results in cell lysis.

Mutations that result in undetectable activity of aspartoacylase, which catalyzes the deacetylation of N-acetyl-L-aspartate, correlate with Canavan Disease, a neurodegenerative disorder usually fatal during childhood. The underlying biochemical mechanisms of how these mutations ablate activity are poorly understood. Therefore, we developed and tested a three-dimensional homology model of aspartoacylase based on zinc dependent carboxypeptidase A. Mutations of the putative zinc-binding residues (H21G, E24D/G, and H116G), the general proton donor (E178A), and mutants designed to switch the order of the zinc-binding residues (H21E/E24H and E24H/H116E) yielded wild-type aspartoacylase protein levels and undetectable ASPA activity. Mutations that affect substrate carboxyl binding (R71N) and transition state stabilization (R63N) also yielded wild-type aspartoacylase protein levels and undetectable aspartoacylase activity. Alanine substitutions of Cys124 and Cys152, residues indicated by homology modeling to be in close proximity and in the proper orientation for disulfide bonding, yielded reduced ASPA protein and activity levels. Finally, expression of several previously tested (E24G, D68A, C152W, E214X, D249V, E285A, and A305E) and untested (H21P, A57T, I143T, P183H, M195R, K213E/G274R, G274R, and F295S) Canavan Disease mutations resulted in undetectable enzyme activity, and only E285A and P183H showed wild-type aspartoacylase protein levels. These results show that aspartoacylase is a member of the caboxypeptidase A family and offer novel explanations for most loss-of-function aspartoacylase mutations associated with Canavan Disease.

Y ersinia pestis is the causative agent of plague, a rare infection in humans that usually appears in the bubonic form due to flea bites. Pneumonic plague is an infection of the respiratory tract which can be contagious and rapidly fatal. This form of plague has an incubation period of 3 to 5 days and a mortality rate near 100% (1, 2). Antibiotic intervention can offer relief but only if started very early in the infection (1). Because of the scattered information on MICs under a variety of nonstandardized testing conditions (3-6) and the lack of comparative data on type strains, we report specific antibiotic susceptibility results from use of the Clinical and Laboratory Standards Institute (CLSI) microdilution broth methodology for 30 strains of Y. pestis. This information will be highly useful as baseline data in the event of wartime or terrorist release and for natural and laboratory-acquired infections. MATERIALS AND METHODSThe Y. pestis strains used in this study (shown in Table 1) were obtained from the USAMRIID collection and selected to represent established biovars, genotypes, and isotypes (7,8). Most of the antibiotics were obtained from the U.S. Pharmacopoeia (Rockville, MD) except for ceftriaxone and fusidic acid (Sigma Chemical Co., St. Louis, Mo), cethromycin (Advanced Life Sciences), telithromycin (Sanofi-Aventis), garenoxacin (ScheringPlough), gemifloxacin (Oscient), ertapenem (Merck), faropenem (Replidyne), and tigecycline (Wyeth). Most of the stock solutions (5 mg/ml) were prepared for each drug in the appropriate solvents, based on the current CLSI recommendations (9), and stored until use at Ϫ70°C. The concentration of our amoxicillin-clavulanate (2:1) stock was 5 mg/2.5 mg per ml, and that of the co-trimoxazole stock was 5 mg/ml sulfamethoxazole and 0.26 mg/ml trimethoprim (19:1). The MICs were determined by the microdilution method in 96-well plates as previously described (10). Antibiotics were serially diluted 2-fold in 50 l of cation-adjusted Mueller-Hinton broth (CAMHB). The antibiotic range in the plates was 64 to 0.008 g/ml based on a final well volume of 100 l after inoculation. The inocula were prepared by picking several colonies from sheep blood agar (SBA) plates grown for 36 to 48 h at 28°C, suspended, and diluted with CAMHB to a bacterial cell density of 10 6 CFU/ml (conversion factor, 5 ϫ