Chemical shift mapping of shikimate-3-phosphate binding to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase.

To facilitate evaluation of enzyme-ligand complexes in solution, we have isolated the 26-kDa N-terminal domain of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase for analysis by NMR spectroscopy. The isolated domain is capable of binding the substrate shikimate-3-phosphate (S3P), and this letter reports the localization of the S3P binding site using chemical shift mapping. Based on the NMR data, we propose that Ser23, Arg27, Ser197, and Tyr200 are directly involved in S3P binding. We also describe changes in the observed nuclear Overhauser effects (NOEs) that are consistent with a partial conformational change in the N-terminal domain upon S3P binding.

Shikimate-3-phosphate binds to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase.

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the transfer of the enolpyruvyl moiety from phosphoenolpyruvate (PEP) to shikimate-3-phosphate (S3P). Mutagenesis and X-ray crystallography data suggest that the active site of the enzyme is in the cleft between its two globular domains; however, they have not defined which residues are responsible for substrate binding and catalysis. Here we attempt to establish the binding of the substrate S3P to the isolated N-terminal domain of EPSP synthase using a combination of NMR spectroscopy and isothermal titration calorimetry. Our experimental results indicate that there is a saturable and stable conformational change in the isolated N-terminal domain upon S3P binding and that the chemical environment of the S3P phosphorus when bound to the isolated domain is very similar to that of S3P bound to EPSP synthase. We also conclude that most of the free energy of S3P binding to EPSP synthase is contributed by the N-terminal domain.

Reconstruction of orbital wall fenestrations with polyglactin 910 film.

Medial orbital wall fenestrations were created bilaterally in 16 adult cats. The fenestrations were reconstructed with polyglactin 910 film, Dacron-reinforced silicone sheeting, or no implant. Polyglactin 910 was found to be well tolerated in this traumatized area of paranasal sinus bone and soft tissue and was totally absorbed in 4 months. Dacron-reinforced silicone sheeting induced a long-standing acute inflammatory reaction in a similar milieu. Partial osseus replacement of the orbital fenestrations occurred in all animals, but it was accompanied by distortion and erosion in apposition to the silicone sheeting. The study does not answer the question of whether orbital contour will be maintained on a long-term basis adjacent to a pneumatized sinus following reconstruction with a bioabsorbable implant.

A comprehensive quality assessment program on the autopsy service.

Many previous studies have established the value of the autopsy in assessing clinical diagnostic accuracy. None has described, however, the use of the autopsy as an ongoing, prospective audit of clinical and pathologic performance. The authors herein outline the process and report the initial results of a comprehensive quality assessment program on their autopsy service. Particular emphasis was placed on evaluation of the responsiveness of the autopsy to clinical questions and interests. The four parts of the overall program included an epidemiologic survey of causes of death in the authors' patient population, a determination of the clinical significance of autopsy findings, an assessment of clinical factors that may have contributed to death, and a quality control mechanism of the autopsy itself. The process can be adapted to a variety of hospital settings.

Hypercalciuria in hyperprolactinemic rats: effects of benzthiazide.

There is evidence that prolactin (PRL) excess plays a role in the etiology of osteoporosis associated with human prolactinoma. Calcium balance in human hyperprolactinemia has not been thoroughly investigated. In the present study, rats with excess circulating PRL levels (male anterior pituitary-grafted Fischer 344 rats) had urinary calcium excretion twice that of control rats (4.16 +/- 0.43 v 2.25 +/- 0.30 mg/24h X 100 g BW). Calcium excretion expressed per mg of calcium intake was also high in pituitary-grafted rats. The excess calcium excretion in hyperprolactinemic rats was not accompanied by a concomitant rise in sodium excretion. This dissociation suggests that PRL has an effect on the renal handling of calcium. Since thiazide diuretics have a well-described hypocalciuric action, their effect was tested in these rats. In normal rats, benzthiazide, a long-acting agent, significantly reduced urinary calcium excretion in a dose-dependent fashion. Hyperprolactinemic rats responded to benzthiazide in a manner similar to control rats. In pituitary-grafted rats, benzthiazide also decreased the calcium excretion to intake ratio and normalized the calcium to sodium excretion ratio. Since the hypercalciuria of experimental hyperprolactinemia can be corrected by thiazide diuretics, these agents may have therapeutic potential in human PRL excess.