13C NMR studies of acetate metabolism during sporulation of Saccharomyces cerevisiae.

The sporulation of Saccharomyces cerevisiae in the presence of [2-13C]acetate was studied by 13C NMR spectroscopy. The fate of 13C label was analyzed in vivo and in cell extracts. During the first 4 hr of sporulation the major metabolite produced from [2-13C]acetate utilization was glutamate. From the labeling pattern observed it is concluded that both the tricarboxylic acid cycle and the glyoxylate cycle are operating. After about 4 hr trehalose is made. Comparison of the doublet/singlet ratios for C-1,1(1) and C-6,6(1) of trehalose shows a steady drop in the ratio of C-1, C-2-coupled species over trehalose labeled only at C-1 in the C-1, 2 segment of the molecule. The negative correlation of this ratio with that for the C-5, 6 segment indicates a cycling of glucose through the hexose monophosphate shunt. Subsequently fatty acid biosynthesis commences. Large amounts of saturated fatty acid were made. There were conspicuous differences observed in the metabolism of [2-13C]acetate between sporulating and vegetatively growing cells.     

Abnormal pattern of gastric emptying of liquid in chronic duodenal ulcer

Gastric emptying was measured in 12 patients with chronic duodenal ulceration and compared with the results from 10 healthy volunteers. The test meal of 300 ml 15% dextrose, labelled with 99mTc-DTPA, was ingested in increments over 6 min. Gamma camera imaging proceeded over 30 min, with a 1-min frame time. A direct correction was applied for the fraction emptying into the small bowel during the ingestion period. Gastric emptying at 6 min was significantly greater in the group with duodenal ulcer (14.4 +/- 2.7% vs. 4.2 +/- 0.9%: mean +/- SEM, p less than 0.01). From this time onwards there were no significant differences in the rates of gastric emptying. These results suggest that chronic duodenal ulcer is associated with an abnormal pattern of gastric emptying of liquid, characterised by an initial rapid phase.     

α-Fluorophosphonates reveal how a phosphomutase conserves transition state conformation over hexose recognition in its two-step reaction

β-Phosphoglucomutase (βPGM) catalyzes isomerization of β-d-glucose 1-phosphate (βG1P) into d-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a β-d-glucose 1,6-bisphosphate (βG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of βG1P deliver novel step 1 transition state analog (TSA) complexes for βPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the β-d-glucopyranose ring in the βG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C)O–P bond orientation for binding the phosphate in the inert phosphate site differs by ∼30° between steps 1 and 2. By contrast, the orientations for the axial O–Mg–O alignment for the TSA of the phosphoryl group in the catalytic site differ by only ∼5°, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of βG16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.Efficient enzyme catalysis of the manipulation of phosphates is one of the great achievements of evolution (1). Enzymes that operate on phosphate monoesters and anhydrides transfer the phosphoryl moiety, PO₃⁻, with rate accelerations approaching 10²¹ for monoesters, placing them among the most proficient of all enzymes (1). Phosphomutases, including α-phosphoglucomutase (αPGM) (2, 3) and β-phosphoglucomutase (βPGM) (4–6), phosphoglycerate mutase (7), α-phosphomannomutase (αPMM/PGM) (8), and N-acetylglucosamine-phosphate mutase (9), merit special attention because these enzymes have to be effective in donating a phosphoryl group to either of two hydroxyl groups that have intrinsically different reactivity. Only when both half-reactions of a phosphomutase are accessible to mechanistic analysis can the problem of how an enzyme accommodates two distinct chemistries within a single active site be resolved. Hexose 1-phosphate mutases, including enzymes central to glycolysis and other metabolic pathways, are well characterized (10, 11). They are generally activated by phosphorylation to form a covalent phosphoenzyme, which then donates its PO₃⁻ group to either of its substrates to deliver a common, transient, hexose 1,6-bisphosphate intermediate species. However, structural studies on phosphomutases are complicated by the rapid and often imbalanced equilibrium position between the substrates, and kinetic studies are problematic because of competitive, parallel pathways of enzyme activation and substrate inhibition (12, 13). As a result, transition states (TSs) for both half-reactions have not hitherto been accessible for mechanistic analysis.βPGM is the best-characterized hexose 1-phosphate mutase and is a member of the haloacid dehalogenase (HAD) superfamily (14), which has 58 HAD homologs in Homo sapiens (11). The key cellular role for βPGM is to support growth on maltose (14), which demands isomerization of β-d-glucose 1-phosphate (βG1P) via β-d-glucose 1,6-bisphosphate (βG16BP) into d-glucose 6-phosphate (G6P), a universal source of cellular energy. This interconversion is achieved via a transient, covalent phosphoenzyme intermediate involving an essential aspartic acid, Asp8, to conserve the phosphoryl group that migrates intermolecularly (Fig. 1). Mechanistically, this pathway demands the architecture of the catalytic site to be effective in promoting phosphoryl transfer from phospho-Asp8 to the 6-OH group of βG1P (step 1), followed by reverse phosphoryl transfer from 1β-OH of βG16BP to Asp8 (step 2).Open in a separate windowFig. 1.Reaction scheme and free energy profile for the conversion of βG1P into G6P via βG16BP catalyzed by βPGM. The phosphoryl transfer reaction between βG1P and the phosphoenzyme (βPGM^P) is step 1 (transferring phosphate is shown in blue), and the equivalent reaction between G6P and the phosphoenzyme is step 2 (transferring phosphate is shown in red). The two intermediate complexes are labeled βG16BP and βG61BP to indicate the two orientations of bound β-bisphosphoglucose. Intramolecular hydrogen bonds within the glucose phosphates are indicated in green. The PDB ID codes (shown in brown) for the structures of metal fluoride ground state analog (GSA) and TSA complexes are listed next to the corresponding steps. G6P is ca. 8 kJ⋅mol⁻¹ lower in free energy than βG1P at equilibrium (12). βG1P binds fivefold less tightly than G6P in an AlF₄⁻ TSA complex, corresponding to a binding energy difference of ca. 4 kJ⋅mol⁻¹. This places the TSA for step 1 (blue) ca. 12 kJ⋅mol⁻¹ (4 kJ⋅mol⁻¹ + 8 kJ⋅mol⁻¹) higher in free energy than the TSA for step 2 (red). The free energy levels of TS1 and TS2 are placed only approximately, using the assumption that the free energy difference (wavy arrows) between the TSA complex and the true TS is similar for both step 1 and step 2. The approximate relative free energy levels for the intermediate enzyme-bound states denoted with βG16BP and βG61BP are based on published data (13).Step 2 has been studied intensively, with analyses focused on structural studies of trifluoromagnesate (MgF₃⁻) and tetrafluoroaluminate (AlF₄⁻) transition state analogs (TSAs) and trifluoroberyllate ground state analogs for G6P complexes (4–6, 15). ¹⁹F NMR resonances for these complexes additionally have provided in situ probes for the electronic and protonic environment of the phosphate moiety in the active site (4–6, 15, 16). Such studies have confirmed a trigonal bipyramidal (tbp) TS associated with inline stereochemistry and general acid–base catalysis, following the rearrangement of near-attack conformers (6). By contrast, step 1, involving phosphorylation of the 6-OH group of βG1P, is not well understood. The corresponding TSA complexes hitherto have proved inaccessible; attempted crystallization of the mutase using βG1P with magnesium and fluoride provides the same MgF₃⁻ TSA complex as is formed directly with G6P because residual enzyme activity catalyzes mutation of βG1P into G6P at a rate competitive with crystallization of the complex (17). Similarly, although ¹⁹F NMR studies have identified a transient TSA complex for an AlF₄⁻ complex of βG1P, it readily isomerizes into the corresponding TSA complex of G6P (SI Appendix, Fig. S1). This impasse is resolved here by the synthesis and use of stable analogs of βG1P that resist mutase-catalyzed isomerization. Because it has been established that α-fluorination of 6-phosphonomethyl-6-deoxy-glucose (G6CP) can enhance or impair analog binding to glucose 6-phosphate dehydrogenase, depending on the stereochemistry of the α-fluorine substituent (18), we have synthesized both diastereoisomeric α-monofluoromethylenephosphonate analogs of βG1P, its methylenephosphonate analog, and the three corresponding phosphonate 1α-hydroxyl analogs. We have identified the two best-binding analogs by ¹⁹F NMR and measured their affinities with βPGM in TSA complexes using fluorescence titration. We have thereby obtained three high-resolution crystal structures of TSA complexes for step 1 of the mutase catalytic reaction. Their comparison with TSA complexes for step 2 establishes the substantially different binding modes for βG1P and G6P in their respective reactions.     

Early experience of MAGEC magnetic growing rods in the treatment of early onset scoliosis

Purpose

Magnetically controlled growing rod systems have been introduced over recent years as an alternative to traditional growing rods for management of early onset scoliosis. The purpose of this paper is to report our early experience of a magnetically controlled growing rod system (MAGEC, Ellipse).

Methods

Review of pre-operative, postoperative and follow-up Cobb angles and spinal growth in case series of eight patients with a minimum 23 months’ follow-up (23–36 months).

Results

A total of six patients had dual rod constructs implanted and two patients received single-rod constructs. Four patients had MAGEC rods as a primary procedure. Four were revisions from other systems. Mean age at surgery in the primary group was 4.5 years (range 3.9–6.9). In patients who had MAGEC as a primary procedure, mean pre-operative Cobb angle was 74° (63–94), with postoperative Cobb angle of 42° (32–56) p ≤ 0.001 (43 % correction). Mean Cobb angle at follow-up was 42° (35–50). Spinal growth rate was 6 mm/year. One sustained proximal screw pull out. A final patient sustained a rod fracture. Mean age at surgery in the revision group was 10.9 years (range 9–12.6). Mean pre-operative Cobb angle was 45° (34–69). Postoperative Cobb angle was 42° (33–63) (2 % correction). Mean Cobb angle at follow-up was 44° (28–67). Mean spinal growth rate was 12 mm/year. Two patients developed loss of distraction.

Conclusion

MAGEC growing rod system effectively controls early onset scoliosis when used as either a primary or revision procedure. Although implant-related complications are not uncommon, the avoidance of multiple surgeries following implantation is beneficial compared with traditional growing rod systems.

     

In Vitro Activity of Plazomicin against 5,015 Gram-Negative and Gram-Positive Clinical Isolates Obtained from Patients in Canadian Hospitals as Part of the CANWARD Study, 2011-2012

Plazomicin is a next-generation aminoglycoside that is not affected by most clinically relevant aminoglycoside-modifying enzymes. The in vitro activities of plazomicin and comparator antimicrobials were evaluated against a collection of 5,015 bacterial isolates obtained from patients in Canadian hospitals between January 2011 and October 2012. Susceptibility testing was performed using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method, with MICs interpreted according to CLSI breakpoints, when available. Plazomicin demonstrated potent in vitro activity against members of the family Enterobacteriaceae, with all species except Proteus mirabilis having an MIC₉₀ of ≤1 μg/ml. Plazomicin was active against aminoglycoside-nonsusceptible Escherichia coli, with MIC₅₀ and MIC₉₀ values identical to those for aminoglycoside-susceptible isolates. Furthermore, plazomicin demonstrated equivalent activities versus extended-spectrum β-lactamase (ESBL)-producing and non-ESBL-producing E. coli and Klebsiella pneumoniae, with 90% of the isolates inhibited by an MIC of ≤1 μg/ml. The MIC₅₀ and MIC₉₀ values for plazomicin against Pseudomonas aeruginosa were 4 μg/ml and 16 μg/ml, respectively, compared with 4 μg/ml and 8 μg/ml, respectively, for amikacin. Plazomicin had an MIC₅₀ of 8 μg/ml and an MIC₉₀ of 32 μg/ml versus 64 multidrug-resistant P. aeruginosa isolates. Plazomicin was active against methicillin-susceptible and methicillin-resistant Staphylococcus aureus, with both having MIC₅₀ and MIC₉₀ values of 0.5 μg/ml and 1 μg/ml, respectively. In summary, plazomicin demonstrated potent in vitro activity against a diverse collection of Gram-negative bacilli and Gram-positive cocci obtained over a large geographic area. These data support further evaluation of plazomicin in the clinical setting.