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Metabolic incorporation of 3H-thymidine into cellular DNA is a widely used protocol to monitor rates of DNA synthesis and cell proliferation. However, this radiochemical has also been reported to induce cell-cycle arrest and apoptosis in addition to DNA damage. Using stable isotope-labeled thymidine, we demonstrate that 3H-thymidine induces dose-dependent inhibition of the rate of DNA synthesis. This inhibition occurred within the first round of replication after addition of the radiolabeled tracer and demonstrates the cytotoxic effects of conventional doses of 3H-thymidine (typically greater than or equal to 1 microCi/ml). These results thus show that stable isotope methods are superior to radioisotopes for determining rates of DNA synthesis and cell replication. Because 3H-thymidine perturbs the very process it was employed to study, experiments using 3H-thymidine to monitor DNA synthesis and cell proliferation should be interpreted with caution.

A remote labeling method has been developed to determine 18 O kinetic isotope effects (KIEs) in Ras-catalyzed GTP hydrolysis. Substrate mixtures consist of 13 C-depleted GTP and [ 18 O, 13 C]GTP that contains 18 O at phosphoryl positions of mechanistic interest and 13 C at all carbon positions of the guanosine moiety. Isotope ratios of the nonvolatile substrates and products are measured by using a chemical reaction interface͞isotope ratio mass spectrometer. The isotope effects are 1.0012 (0.0026) in the ␥ nonbridge oxygens, 1.0194 (0.0025) in the leaving group oxygens (the ␤-␥ oxygen and the two ␤ nonbridge oxygens), and 1.0105 (0.0016) in the two ␤ nonbridge oxygens. The KIE in the ␤-␥ bridge oxygen was computed to be 1.0116 or 1.0088 by two different methods. The significant KIE in the leaving group reveals that chemistry is largely rate-limiting whereas the KIEs in the ␥ nonbridge oxygens and the leaving group indicate a loose transition state that approaches a metaphosphate. The KIE in the two ␤ nonbridge oxygens is roughly equal to that in the ␤-␥ bridge oxygen. This indicates that, in the transition state, Ras shifts one-half of the negative charge that arises from P ␥-O␤-␥ fission from the ␤-␥ bridge oxygen to the two ␤ nonbridge oxygens. The KIE effects, interpreted in light of structural and spectroscopic data, suggest that Ras promotes a loose transition state by stabilizing negative charge in the ␤-␥ bridge and ␤ nonbridge oxygens of GTP. R as is the prototypical member of the family of small G proteins, which along with G␣ subunits of heterotrimeric G proteins, constitute a class of GTP hydrolases that regulate diverse signaling pathways in eukaryotes (1). Ras orchestrates multiple signaling pathways and regulates cell differentiation, proliferation, and apoptosis (2-4). The GTP-bound forms of G proteins are functionally active: that is, they bind to ''effector'' molecules and regulate their activities or location within the cell. Hydrolysis of GTP results in deactivation and effector release (5). In the absence of other factors, the duration of the active signaling state depends on the intrinsic hydrolytic rate of the G protein, which is typically very slow. However, Ras and other G proteins are subject to specific regulation by GTPase-activating proteins (GAPs), which accelerate intrinsic hydrolytic rates by factors ranging from 10 to 10 5 . In particular, RasGAP increases the GTPase rate of Ras by a factor of 10 5 , from 10 Ϫ4 s Ϫ1 to 10 s Ϫ1 (6). Mutations that impair either intrinsic or GAP-facilitated GTPase activity leave Ras in a prolonged state of activation, which is responsible for its role in oncogenic diseases (7).Ras catalyzes the in-line attack of water on the ␥ phosphate of GTP with inversion of configuration (8). However, the nature of the transition state and the rate-limiting step of Ras-catalyzed GTP hydrolysis remain unclear (9-16). A phosphoryl transfer reaction may either proceed through a metaphosphate or a phosphorane intermediate, or by a concerted pathway (Fig. 1) (17, 18...

New and improved strategies are eagerly sought for the rapid identification of microorganisms, particularly in mixtures. Mass spectrometry remains a powerful tool for this purpose. Small acid-soluble proteins (SASPs), which are relatively abundant in Bacillus spores, represent potential biomarkers for species characterization. Despite sharing extensive sequence homology, these proteins differ sufficiently in sequence for discrimination between species. This work focuses on the differences in sequence between SASPs from various Bacillus species. Compilation of SASP sequences from protein database searches, followed by in silico trypsin digestion and analysis of the resulting fragments, identified several species-specific peptides that could be targeted for analysis using mass spectrometry. This strategy was tested and found to be successful in the characterization of Bacillus spores both from individual species and in mixtures. Analysis was performed using an ion trap mass spectrometer with an atmospheric pressure MALDI source. This instrumentation offers the advantage of increased speed of analysis and accurate precursor ion selection for tandem mass spectrometric analysis compared with vacuum matrix-assisted laser desorption/ionization and time-of-flight instruments. The identification and targeting of species-specific peptides using this type of instrumentation offers a rapid, efficient strategy for the identification of Bacillus spores and can potentially be applied to different microorganisms.

BaciUus anthracis and BaciUus cereus are closely related pathogenic organisms that are difficult to differentiate phenotypically or genotypically. It is well known that vegetative and spore forms of bacilli are quite distinct both morphologically and chemically, but spore-specific chemical markers allowing these species to be distinguished have not been previously described. By using gas chromatography-mass spectrometry, vegetative cells and spores of the two species were shown to exhibit distinct carbohydrate profiles. Profiles of vegetative B. anthracis typically contained high levels of galactose but did not contain galactosamine, whereas B. cereus contained galactosamine and generally low levels of galactose. Spore cultures exhibited unique carbohydrate profiles compared with those of vegetative cultures. B. anthracis spore profiles contained rhamnose alone, whereas B. cereus spore profiles contained rhamnose and fucose. Additionally, two spore-specific 0-methylated methylpentoses were discovered. Both B. anthracis and B. cereus spores contained 3-0-methyl rhamnose, whereas B. cereus spores also contained 2-0-methyl rhamnose. Carbohydrate profiling is demonstrated to be a powerful tool for differentiating the two closely related species. Differentiation does not depend on whether organisms are in the vegetative or spore stage of growth.

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Muramic acid is an amino sugar found in eubacterial cell walls and not elsewhere in nature. This study explored the use of electrospray tandem mass spectrometry (ESI MS/MS) in analysis of underivatized muramic acid in bacterial hydrolysates. Fungal hydrolysates were used as negative controls. The only processing used was hydrolysis in sulfuric acid followed by extraction with an organic base (N,N-dioctylmethylamine) to remove the acid prior to ESI MS/MS analysis. Compared with pure muramic acid, bacterial hydrolysates produced more complex ESI mass spectra, such that the protonated molecular ion at m/z 252 was barely detectable. In contrast, product ion spectra of m/z 252 were identical among pure muramic acid, Gram positive bacteria, and Gram negative bacteria. However, no characteristic product ion spectrum was manifested from m/z 252 in fungal samples. This allowed ready, visual differentiation of bacteria and fungi. Multiple reaction monitoring (MRM) following muramic acid fragmentations (m/z 252-->144 and m/z 252-->126) increased sensitivity and allowed quantitative differentiation when compared with the MRM of the internal standard N-methyl-D-glucamine (m/z 196-->44). ESI MS/MS required minimal sample preparation and allowed rapid sample throughput for analysis of muramic acid in whole bacterial cell hydrolysates.