Asthma clinical pathway: an interdisciplinary approach to implementation in the inpatient setting.

Asthma is a leading cause of admission to the pediatric inpatient setting. Despite advances in the treatment of this chronic condition, morbidity and mortality continue to increase. It is also a source of significant variation in clinical practice and redundancy of care elements across various disciplines involved in the management of patients with asthma. A clinical pathway was developed and implemented by a multidisciplinary team at The Children's Hospital of Philadelphia. The unique approach used to strategize implementation combined the expertise of registered nurses, respiratory therapists, medical staff, and case managers and was a significant factor in the pathway's ultimate success. The result was a more standardized and efficient approach to care. Outcome measurements revealed decreased length of stay with no increase in the re-admission rate and cost savings.

Structure-based design of substituted diphenyl sulfones and sulfoxides as lipophilic inhibitors of thymidylate synthase.

Six new diphenyl sulfoxide and five new diphenyl sulfones were designed, synthesized, and tested for their inhibition of human and Escherichia coli thymidylate synthase (TS) and of the growth of cells in tissue culture. The best sulfoxide inhibitor of human TS was 3-chloro-N-((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)-4- (phenylsulfinyl)-N-(prop-2-ynyl)-aniline (7c) that had a Ki of 27 nM. No sulfone improved on TS inhibition by the previously reported 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2- ynylamino)phenyl phenyl sulfone (Ki = 12 nM). Nevertheless, one sulfone, 4-((2-chlorophenyl)sulfonyl)-N-((3,4-dihydro-2-methyl-4-oxo-6- quinazolinyl)methyl)-N-(prop-2-ynyl)aniline, was selected, on the basis of its inhibition of both TS and cell growth, for antitumor testing; it gave a 61% increase in life span to mice bearing the thymidino kinase-deficient L5178Y (TK-) lymphoma. A crystal structure of N-((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)-4-((2- methylphenyl)sulfinyl)-N-(prop-2-ynyl)aniline complexed with E. coli TS was solved and revealed selective binding of one sulfoxide enantiomer. AMBER calculations showed that the enantioselection was due to asymmetric electrostatic effects at the mouth of the active site. In contrast, a similar crystal structure of the sulfoxide 7c, along with AMBER calculations, indicated that both enantiomers bound, but with different affinities. The side chain of Phe176 shifted in order to structurally accommodate the chlorine of the more weakly bound enantiomer.

Structure-based design of lipophilic quinazoline inhibitors of thymidylate synthase.

To develop novel lipophilic thymidylate synthase (TS) inhibitors, the X-ray structure of Escherichia coli TS in ternary complex with FdUMP and the inhibitor 10-propargyl-5,8-dideazafolic acid (CB3717) was used as a basis for structure-based design. A total of 31 novel lipophilic TS inhibitors, lacking a glutamate residue, were synthesized; 26 of them had in common a N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylaniline+ ++ structure in which the aniline was appropriately substituted with simple lipophilic substituents either in position 3 or 4, or in both. Compounds were tested for their inhibition of E. coli TS and human TS and also for their inhibition of the growth in tissue culture of a murine leukemia, a human leukemia, and a thymidine kinase-deficient human adenocarcinoma. The crystal structures of five inhibitors complexed with E. coli TS were determined. Five main conclusions are drawn from this study. (i) A 3-substituent such as CF(3), iodo, or ethynyl enhances binding by up to 1 order of magnitude and in the case of CF(3) was proven to fill a nearby pocket in the enzyme. (ii) A simple strongly electron-withdrawing substituent such as NO(2) or CF(3)SO(2) in the 4-position enhances binding by 2 orders of magnitude; it is hypothesized that the transannular dipole so induced interacts favorably with the protein. (iii) Attempts to combine the enhancements of i and ii in the same molecule were generally unsuccessful (iv) A 4-C(6)H(5)SO(2) substituent provided both electron withdrawal and a van der Waal's interaction of the phenyl group with a hydrophobic surface at the mouth of the active site. The inhibition (K(is) = 12 nM) of human TS by this compound, 7n, showed that C(6)H(5)SO(2) provided virtually as much binding affinity as the CO-glutamate which it had replaced. (v) The series of compounds were poorly water soluble, and also the potent TS inhibition shown by several of them did not translate into good cytotoxicity. Compounds with large cyclic groups linked to position 4 by an SO or SO(2) group did, however, have IC(50)'s in the range 1-5 microM. Of these, 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylamino )phenyl phenyl sulfone, 7n, had IC(50)'s of about 1 microM and was chosen for further elaboration.

Synthesis and biological evaluation of novel 2,6-diaminobenz[cd]indole inhibitors of thymidylate synthase using the protein structure as a guide.

The design, synthesis, and biochemical and biological evaluations of a novel series of 2,6-diaminobenz[cd]indole-containing inhibitors of human thymidylate synthase (TS) are described. The compounds are characterized by having either a pyridine or pyridazine ring in place of the (phenylsulfonyl)morpholinyl group of the known inhibitor N6-[4-(morpholinosulfonyl)benzyl]-N6-methyl-2,6-diaminobenz[ cd]indole glucuronate (i). Active compounds from this series showed human TS inhibition constants below the 10 nM level and were potent, selective submicromolar antitumor agents in cell culture. The compounds were synthesized by reductive alkylation of a substituted 6-aminobenz[cd]indole or reductive cyclization of a substituted 1-cyano-8-nitronaphthalene.

Multiple protein-aspartate phosphatases provide a mechanism for the integration of diverse signals in the control of development in B. subtilis.

The initiation of sporulation in B. subtilis is regulated by the Spo0A transcription factor, which is activated by phosphorylation to control developmental switching from the vegetative to the sporulation state. The level of phosphorylation of Spo0A is regulated by the phosphorelay, a signal transduction system based on the protein-histidine kinase-response regulator two-component paradigm. To initiate sporulation, the cell must recognize and interpret a large variety of environmental, metabolic, and cell cycle signals that influence the phosphorylation level of Spo0A. We describe here a family of protein-aspartate phosphatases with activity on Spo0F approximately P, a response regulator component of the phosphorelay, that provide a mechanism for signal recognition and interpretation. These phosphatases function to drain the phosphorelay, lower Spo0A approximately P levels, and prevent sporulation. The integration of diverse environmental signals that affect the initiation of sporulation likely occurs through the competition between opposing activities of protein kinases and protein phosphatases.

Biochemical characterization of the essential GTP-binding protein Obg of Bacillus subtilis.

An essential guanine nucleotide-binding protein, Obg, of Bacillus subtilis has been characterized with respect to its enzymatic activity for GTP. The protein was seen to hydrolyze GTP with a Km of 5.4 microM and a kcat of 0.0061 min-1 at 37 degrees C. GDP was a competitive inhibitor of this hydrolysis, with an inhibition constant of 1.7 microM at 37 degrees C. The dissociation constant for GDP from the Obg protein was 0.5 microM at 4 degrees C and was estimated to be 1.3 microM at 37 degrees C. Approximately 80% of the purified protein was capable of binding GDP. In addition to hydrolysis of GTP, Obg was seen to autophosphorylate with this substrate. Subsequent release of the covalent phosphate proceeds at too slow a rate to account for the overall rate of GTP hydrolysis, indicating that in vitro hydrolysis does not proceed via the observed phosphoamidate intermediate. It was speculated that the phosphorylated form of the enzyme may represent either a switched-on or a switched-off configuration, either of which may be normally induced by an effector molecule. This enzyme from a temperature-sensitive mutant of Obg did not show significantly altered GTPase activity at the nonpermissive temperature.

Structure of and kinetic channelling in bifunctional dihydrofolate reductase-thymidylate synthase.

The bifunctional enzyme dihydrofolate reductase-thymidylate synthase catalyses both the reductive methylation of 2'-deoxyuridylate and the subsequent reduction of dihydrofolate to yield 2'-deoxythymidylate and tetrahydrofolate at two spacially discrete sites situated on different protein domains. The X-ray structure of dihydrofolate reductase-thymidylate synthase from Leishmania major indicates that transfer of dihydrofolate between these sites does not occur by transient binding at both sites but rather by movement of dihydrofolate across the surface of the protein. The enzyme has an unusual surface charge distribution that could account for this channelling of dihydrofolate between active sites.

Design of thymidylate synthase inhibitors using protein crystal structures: the synthesis and biological evaluation of a novel class of 5-substituted quinazolinones.

The design, synthesis, and biological evaluation of a new class of inhibitors of thymidylate synthase (TS) is described. The molecular design was carried out by a repetitive crystallographic analysis of protein-ligand structures. At the onset of this project, we focused on the folate cofactor binding site of a high-resolution ternary crystal complex of Escherichia coli TS, 5'-fluorodeoxyuridylate (5-FdUMP) and a classical glutamate-containing folic acid analog. A preliminary ternary crystal structure of a novel compound was successfully solved. Upon analysis of this initial complex, further structural elaborations were made, and a series of active 5-(arylthio)quinazolinones was developed. The synthetic strategy was based on the displacement of a halogen at the 5-position of a quinazolinone by various aryl thioanions. The compounds were tested for inhibition of purified E. coli and/or human TS, and were assayed for cytotoxicity against three tumor cell lines in vitro. Significant thymidine protection effects were observed with several of the inhibitors, indicating that TS was the intracellular locus of activity.

Design and synthesis of novel 6,7-imidazotetrahydroquinoline inhibitors of thymidylate synthase using iterative protein crystal structure analysis.

Antifolate inhibitors of thymidylate synthase (TS) have primarily been based on the structure of folic acid. This paper describes the identification and development of novel 6,7-imidazotetrahydroquinoline TS inhibitors by iterative ligand design, synthesis, and crystallographic analysis of protein-inhibitor complexes. Beginning with a high-resolution crystal structure of E. coli TS (TS, EC 2.1.1.45), an imidazotetrahydroquinoline inhibitor was designed de novo to occupy the folate binding pocket. Structural modifications of the initial compound 1h (Ki approximately 5 microM human/E. coli TS) were then made on the basis of feedback from additional cocrystal structures and activity data. An amino group in the 2-position of the imidazole was found to increase the potency of the series by 1-2 orders of magnitude. Other substitutions on the imidazole ring (1-CH3, 2-CH3, 2-NHCH3, 2-SCH3) generally led to weaker inhibition. Additional improvements in activity were obtained by modification of the substituents on the tetrahydroquinoline nitrogen, bringing the Ki of three of the compounds below 15 nM against the human TS enzyme. The compounds were tested for cytotoxicity and were shown to inhibit the growth of three tumor cell lines in vitro.

Restrictive dermopathy. Report of two affected siblings and a review of the literature.

BACKGROUND: Restrictive dermopathy is a lethal genetic disorder consisting of abnormally tight skin, generalized joint contractures, distinctive facies, and pulmonary hypoplasia. Autosomal recessive inheritance has been suggested based on multiply affected siblings and some reports of parental consanguinity. This article describes two siblings with the restrictive dermopathy syndrome and reviews previously reported cases. OBSERVATIONS: Eight other cases have been reported in the literature as restrictive dermopathy. These cases have shared striking similarities in their clinical histories and phenotypes. The skin in these infants has been described as rigid and tense, with skin biopsy specimens showing a thick epidermis, thin dermis, abnormally arranged collagen bundles, and poorly developed appendages. Other prominent features are flexion contractures and craniofacial and pulmonary abnormalities. The genetic and/or developmental defects leading to the restrictive dermopathy syndrome are presently not known. CONCLUSIONS: The restrictive dermopathy syndrome is distinct and is easily differentiated from other congenital diseases such as the icthyoses and also from the clinical conditions of sclerema neonatorum and subcutaneous fat necrosis of the newborn. Recognition of this syndrome is important for determining the prognosis of affected infants and for recommending genetic counseling to affected families.

Crystal-structure-based design and synthesis of benz[cd]indole-containing inhibitors of thymidylate synthase.

The X-ray crystal-structure-based design, synthesis, and biological activity of a novel family of benz[cd]indole-containing inhibitors of thymidylate synthase (TS) are described. The structure-activity of the lead compound was studied by conceptually dividing the molecule into four regions and independently optimizing the substituents for each region. Combination of favored substituents for each region led to inhibitors with Ki's against the human enzyme in the range of 10-20 nM. Thymidine shift experiments suggested that the cytotoxic properties of the best enzyme inhibitors were due to TS targeting in cells. The inhibitors were synthesized from substituted 6-aminobenz[cd]indol-2(1H)-ones by alkylation with both a simple alkyl group and a substituted benzylic portion. The 2,6-diaminobenz[cd]indoles were prepared from the corresponding lactams by conversion to the thiolactam, alkylation to the methylated thiolactam, and then displacement with a substituted or unsubstituted amine.

The effect of recombinant human growth hormone on wound healing in normal individuals.

Growth hormone therapy has been suggested to speed wound healing in postoperative patients and in patients with severe burns. This study was undertaken to determine the effect of recombinant human growth hormone on the rate of wound healing in normal individuals. Twenty-three healthy males were evaluated in a randomized, double-blind placebo-controlled study. Each subject received a split-thickness wound (Davol-Keratome) on one buttock and a full-thickness wound (3-mm punch biopsy) on the other. The full-thickness wound healed significantly more slowly in the recombinant human growth hormone-treated group as compared with the placebo control group (t-test, P = .001). No statistically significant difference was noted in the healing of the split-thickness wounds. It is concluded that recombinant human growth hormone may impede healing in normal patients with full-thickness wounds as compared with treatment with placebo. We cannot rule out, however, that the recombinant human growth hormone affected the quality of the scab in full-thickness wounds and thereby only appeared to alter the wound-healing process.

Alteration of the metastatic potential of line 1 lung carcinoma cells: opposite effects of class I antigen induction by interferons versus DMSO or gene transfection.

Class I antigens are necessary for the recognition of tumor cells by cytotoxic T lymphocytes (CTL). The line 1 lung carcinoma is a spontaneous murine tumor deficient in class I antigen expression. Consistent with this, line 1 cells are highly metastatic in vivo. We investigated whether increasing class I antigen expression on line 1 cells could alter the metastatic potential of these tumor cells using an in vivo lung metastasis model. We used three methods to induce class I antigen expression on line 1 cells: gene transfection, treatment with dimethyl sulfoxide (DMSO), or treatment with interferon (IFN)-beta or -gamma. We found that line 1 cells expressing a transfected class I gene were significantly less metastatic than parental line 1 cells. DMSO-treated line 1 cells also formed significantly fewer metastases than parental line 1 cells. These results indicate that increased class I antigen expression decreases the metastatic potential of line 1 cells in vivo. However, we did not observe a significant decrease in the number of lung metastases in mice receiving line 1 cells treated with IFN-beta or -gamma, despite high levels of class I antigen expression. Thus, increasing class I antigen expression with IFN has an opposite effect on metastasis from class I antigen expression induced by transfection or DMSO. These results show that the method used to increase class I antigen expression is critical in terms of the in vivo effect observed. To investigate a possible mechanism for the differences observed in vivo between these class I expressing cells, we tested whether IFN alters or blocks susceptibility of line 1 cells to immune effector cells. We found IFN treatment increased the ability of line 1 cells to be recognized by CTL but concomitantly decreased the susceptibility of line 1 cells to NK cell lysis by a non-class I antigen-related mechanism. In contrast, transfected or DMSO-treated line 1 cells which were less metastatic in vivo were susceptible to both CTL and NK-mediated lysis. Taken together, these results suggest that immune intervention against metastasizing line 1 cells may involve NK cells and CTL.

Isolation and characterization of the Escherichia coli mutL gene product.

The Escherichia coli mutL gene product has been purified to near homogeneity from an overproducing clone. The mutL locus encodes a polypeptide of 70,000 daltons as determined by denaturing gel electrophoresis. The native molecular weight of MutL protein as calculated from the sedimentation coefficient of 5.5 S and Stokes radius of 61 A is 139,000 daltons, indicating that MutL exists as a dimer in solution. In addition to its ability to complement methyl-directed DNA mismatch repair in mutL-deficient cell-free extracts, DNase I protection experiments demonstrate that the purified MutL protein interacts with the MutS-heteroduplex DNA complex in the presence of ATP.

Isolation and characterization of the Escherichia coli mutH gene product.

The Escherichia coli mutH gene product has been isolated in near homogeneous form using an in vitro complementation assay for DNA mismatch correction (Lu, A.-L., Clark, S., and Modrich, P. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4639-4643) which is dependent on mutH function. The protein has a subunit Mr of 25,000, and purified preparations contain a Mg2+-dependent endonuclease activity which cleaves 5' to the dG of d(GATC) sequences to generate 5'-phosphoryl and 3'-hydroxyl termini. Symmetrically methylated d(GATC) sites are resistant to the endonuclease, hemimethylated sequences are cleaved on the unmethylated strand, and unmethylated d(GATC) sites are usually subject to scission on only one DNA strand. Although this endonuclease activity is extremely weak (less than 1 scission/h/mutH monomer equivalent) and cleavage at a d(GATC) site does not depend on the presence of a mismatched base pair within the DNA substrate, the activity does not appear to be a contaminant of mutH preparations. d(GATC) endonuclease activity and mutH complementing activity co-purify through multiple column steps without change in relative specific activities, and both activities co-electrophorese under native conditions. These findings suggest that the mutH product functions at the strand discrimination stage of mismatch correction and that this stage of the reaction involves scission of the unmethylated DNA strand.

Yeast inorganic pyrophosphatase. A model for active-site structure based on 113Cd2+ and 31P NMR studies.

Equilibrium dialysis and 113Cd2+ NMR studies in the presence of inorganic phosphate (Pi) provide clear evidence for the existence of four well-defined Cd2+ sites per yeast inorganic pyrophosphatase subunit. Parallel 31P NMR studies demonstrate the existence of two binding sites per subunit for Pi and provide strong confirmatory evidence for a small amount of enzyme-bound inorganic pyrophosphate in equilibrium with enzyme-bound Pi. Such inorganic pyrophosphate formation was demonstrated by chemical analysis earlier [Welsh, K.M., Armitage, I.M., & Cooperman, B.S. (1983) Biochemistry 22, 1046-1054]. In this same earlier paper, we provided evidence for inner-sphere contact between enzyme-bound Cd2+ and Pi bound in the higher affinity of the two Pi sites. We now present heteronuclear decoupling evidence that one to three different Cd2+ ions make such contact. The divalent metal ion cofactor conferring the highest activity on inorganic pyrophosphatase is Mg2+, and we present evidence from competition experiments that such inner-sphere contact is also likely for the Mg2+-enzyme. On the other hand, these experiments also show that some metal ion binding sites on the enzyme bind Mg2+ and not Cd2+, and some bind Cd2+ and not Mg2+. These results are considered along with others obtained recently in proposing an active-site structure for inorganic pyrophosphatase.

Evidence that catalysis by yeast inorganic pyrophosphatase proceeds by direct phosphoryl transfer to water and not via a phosphoryl enzyme intermediate.

In this work, we show that adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) is a substrate for yeast inorganic pyrophosphatase (PPase) (EC 3.6.1.1) and further, using chirally labeled [gamma-17O,18O]ATP gamma S, that enzyme-catalyzed hydrolysis to produce chiral inorganic thio[17O,18O]phosphate proceeds with inversion of configuration. Both the synthesis of chiral ATP gamma S and the determination of inorganic thiophosphate configuration were carried out as described by Webb [Webb, M. R. (1982) Methods Enzymol. 87, 301-316]. We also show in a single turnover experiment performed in H2(18)O that 1 mol each of 18O16O3P and 16O4P is produced per mol of inorganic pyrophosphate hydrolyzed, a strong indication that oxygen uptake to form inorganic phosphate on PPase catalysis of inorganic pyrophosphate hydrolysis comes directly from H2O. These two results provide strong evidence for the conclusion that PPase catalyzes inorganic pyrophosphate hydrolysis via a single-step direct phosphoryl transfer to water and does not involve formation of a phosphorylated enzyme intermediate.

Thermodynamics, kinetics, and mechanism in yeast inorganic pyrophosphatase catalysis of inorganic pyrophosphate: inorganic phosphate equilibration.

We have developed two methods for quantitatively measuring inorganic pyrophosphate (PPi) in the presence of 10(3)--10(4) molar excesses of inorganic phosphate (Pi) and used them to measure the extent of enzyme-bound pyrophosphate (EPPi) formation in solutions of yeast inorganic pyrophosphatase and Pi. We have also measured the rate of enzyme-catalyzed H2O--phosphate oxygen exchange. We find both processes to have essentially identical dependence on Mg2+ and Pi concentrations, thus providing important confirmation for the recent proposal by Janson et al. (1979) that oxygen exchange proceeds via EPPi formation. Our results are consistent with a model in which three Mg2+ per active site are required for EPPi formation but inconsistent with a model requiring only two Mg2+ per active site and permit the formulation of an overall scheme for inorganic pyrophosphatase catalysis of PPi--Pi equilibration as well as the evaluation of equilibrium and rate constants in this scheme. The major results and conclusions of our work are the following: (a) the equilibrium constant for PPi (enzyme-bound) in equilibrium with 2Pi (enzyme-bound) is 4.8; (b) following PPi hydrolysis, the first released Pi contains an oxygen from solvent water; (c) the steps for PPi hydrolysis on the enzyme and for release of both product Pi's are all partially rate determining in overall enzyme-catalyzed PPi hydrolysis; (d) PPi formation on the enzyme is rate determining for H2O--Pi oxygen exchange; (e) PPi dissociation from the enzyme is very slow and is the rate-determining step in Pi--PPi exchange (Cohn, 1958; Janson et al., 1979). This also accounts for the observation that the calculated dissociation constant for MgPPi complex binding to enzyme is considerably lower than the measured Km for enzyme-catalyzed MgPPi hydrolysis.