Probing the roles of active site residues in phosphatidylinositol-specific phospholipase C from Bacillus cereus by site-directed mutagenesis

The role of amino acid residues located in the active site pocket of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus[Heinz, D. W., Ryan, M., Bullock, T., & Griffith, O. H. (1995) EMBO J. 14, 3855-3863] was investigated by site-directed mutagenesis, kinetics, and crystal structure analysis. Twelve residues involved in catalysis and substrate binding (His32, Arg69, His82, Gly83, Lys115, Glu117, Arg163, Trp178, Asp180, Asp198, Tyr200, and Asp274) were individually replaced by 1-3 other amino acids, resulting in a total number of 21 mutants. Replacements in the mutants H32A, H32L, R69A, R69E, R69K, H82A, H82L, E117K, R163I, D198A, D198E, D198S, Y200S, and D274S caused essentially complete inactivation of the enzyme. The remaining mutants (G83S, K115E, R163K, W178Y, D180S, Y200F, and D274N) exhibited reduced activities up to 57% when compared with wild-type PI-PLC. Crystal structures determined at a resolution ranging from 2.0 to 2.7 A for six mutants (H32A, H32L, R163K, D198E, D274N, and D274S) showed that significant changes were confined to the site of the respective mutation without perturbation of the rest of the structure. Only in mutant D198E do the side chains of two neighboring arginine residues move across the inositol binding pocket toward the newly introduced glutamic acid. An analysis of these structure-function relationships provides new insight into the catalytic mechanism, and suggests a molecular explanation of some of the substrate stereospecificity and inhibitor binding data available for this enzyme.     

The functional organization of the adaptive and compensatory reactions of the endocrine glands

Current concepts of the systemic organization and interrelationship between adaptive and compensatory responses of the endocrine glands and hormone-producing cells are reviewed. Emphasis is made on the involvement of prolactin, insulin endorphins and other neuropeptides into stress responses. A problem of the multihormonal control of physiological functions and role of blood protein carriers and hormonal cytoreceptors in pathogenesis of various diseases are discussed. Special attention is paid to the importance of endocrine function reserves testing for diagnosis of premorbid states and for estimation of human psychophysiological and physical potentials.     

Fulminant cytomegalovirus colitis without prior immune deficiency or colonic disease

Fulminant primary Cytomegalovirus (CMV) colitis can occur in immunocompetent individuals and mimics inflammatory bowel disease. Cytomegalovirus inclusions are found in rectal or colonic biopsy specimens. Thus, careful histological evaluation of mucosa biopsies is essential for the diagnosis of this entity.     

pH Dependence of Complex Formation Between Condensed Tannins and Proteins

The interaction between condensed grape tannins and some proteins was studied turbidimetrically. Tannins precipitated protein efficiently at pH values up to the isoelectric point of the individual protein. At slightly higher pH values there was a sharp decrease in complex formation. This “critical pH” was usually observed within approximately 0.5 pH unit of the isoelectric point. A close correlation (r = 0.96) was obtained between the isoelectric point and the critical pH of proteins. The effects of pH and ionic strength on turbidity formation by tannins and bovine serum albumin showed that an increasing salt concentration tended to increase turbidity at pH 3, but it had no significant effect at pH 4. In contrast at pH 5 high salt concentration tended to decrease turbidity. Employing an ultrafiltration technique, the binding behavior of interacting tannin and protein was described by a typical Brunauer type II adsorption curve.     

Intracellular glucose switches between cyclic ADP-ribose and inositol trisphosphate triggering of cytosolic Ca2+ spiking

Cyclic ADP-ribose (cADPR) is a potentially important intracellular Ca2+ releasing messenger [1-5]. In pancreatic acinar cells where intracellular infusion of both inositol trisphosphate (IP3) and cADPR evoke repetitive Ca2+ spiking [6], the cADPR antagonist 8-NH2-cADPR [7], which blocks cADPR-evoked but not IP3-evoked Ca2+ spiking, can abolish Ca2+ spiking induced by physiological levels of the peptide hormone cholecystokinin (CCK) [8]. We have tested the effect of intracellular glucose on the ability of IP3, cADPR and CCK to induce cytosolic Ca2+ spikes in pancreatic acinar cells. In order to gain access to the intracellular cytosol, we used the whole-cell configuration of the patch-clamp technique [9] and monitored cytosolic Ca2+ concentration changes by measuring the Ca(2+)-dependent ionic current [10-13]. Glucose (300 microM to 10 mM) in the patch pipette/intracellular solution prevented cADPR from evoking Ca2+ spiking. The same effect was observed with 2-deoxy-glucose, but not L-glucose. In contrast, glucose potentiated IP3-evoked Ca2+ spiking. CCK evoked Ca2+ spiking irrespective of the presence or absence of intracellular glucose, but the cADPR antagonist 8-NH2-cADPR blocked CCK-evoked Ca2+ spiking only in the absence of intracellular glucose. This suggests that the hormone can evoke Ca2+ spiking via either the IP3 or the cADPR pathway. The intracellular glucose level may control a switch between these two pathways.     

Sestamibi scanning is inadequate for directing unilateral neck exploration for first-time parathyroidectomy

OBJECTIVE: To determine whether technetium Tc 99m sestamibi scanning is accurate enough to allow surgeons to perform unilateral neck exploration for first-time parathyroidectomy in patients with primary hyperparathyroidism. DESIGN: Retrospective review. SETTING: University tertiary care center. PATIENTS: Forty patients with primary hyperparathyroidism who underwent sestamibi scanning before first-time parathyroidectomy, of whom 28 had single adenomas, 9 had multiple adenomas, and 3 had hyperplasia. INTERVENTIONS: All 40 patients underwent bilateral neck exploration with identification of 4 parathyroid glands. MAIN OUTCOME MEASURES: We compared the results of preoperative sestamibi scanning with operative and histologic findings. We then used these data to calculate the projected success rates of parathyroidectomy if unilateral neck explorations had been performed based on the results of sestamibi scanning, instead of bilateral explorations. RESULTS: Sestamibi scanning was correct in 20 (71%) of 28 patients with single adenomas, 4 (44%) of 9 patients with multiple adenomas, and 0 (0%) of 3 patients with hyperplasia. If unilateral neck explorations had been performed on the basis of localization by sestamibi scanning, parathyroidectomy would have failed in 4 (10%) of 40 patients. CONCLUSIONS: Sestamibi scanning, although helpful, is inadequate for directing unilateral neck exploration for first-time parathyroidectomy. Surgeons who perform unilateral neck exploration based on the results of sestamibi scanning will record a higher failure rate and incur higher costs than those who perform bilateral neck exploration for first-time parathyroidectomy.     

Mechanical circulatory support in children

Nine children (aged 1.2-15 years) have been treated with mechanical circulatory support devices at our institution. Indications for treatment were acute cardiac allograft rejection (n = 4), postcardiotomy cardiogenic shock (n = 4), and bridge to cardiac transplantation (n = 1). Eight patients required left ventricular support, and one required biventricular support. A BioMedicus centrifugal pump was used in eight patients, and a Hemopump intra-aortic axial flow device was used in one patient. In two patients, an intra-aortic balloon pump was in place at the time that circulatory support was instituted. Mechanical support time ranged from 2 to 139 h, and the average flow index was 2.31 l/min per m2. Three patients required hemodialysis during support, and one patient required re-exploration because of mediastinal hemorrhage. Recovery of native ventricular function was assessed by transthoracic or transesophageal echocardiography, and weaning from the device was achieved by gradually decreasing pump flow in increments of 0.1 to 0.5 l/min. Seven patients were successfully weaned from support. Two hospital deaths occurred after circulatory support had been discontinued: one patient died of respiratory failure and the other of gram-negative pneumonia and sepsis. The five surviving patients experienced no significant complications, and their hemodynamic indices were normal at the time of discharge. At a mean follow-up of 28.8 months, these patients are leading active unrestricted lives, with no long-term device-related sequelae. Based on this experience, mechanical circulatory support is feasible in children who experience profound circulatory failure from a variety of causes.