If you build it, will they come? Using a mobile safety centre to disseminate safety information and products to low-income urban families.

OBJECTIVE: To examine the utilisation-related outcomes associated with visiting the Johns Hopkins CareS (Children are Safe) Mobile Safety Center (MSC), a 40-foot vehicle designed to deliver effective injury prevention interventions and education to low-income urban families. DESIGN AND SETTING: Utilisation-related data were collected when the MSC was accessible at a community health centre and at community events from August 2004 to July 2006 in Baltimore City. SUBJECTS: Adults bringing their child for well child care at a community health centre and MSC visitors at community events. INTERVENTIONS: Low-cost safety products and free personalized educational services are provided on the MSC, which replicates a home environment and contains interactive exhibits. MAIN OUTCOME MEASURES: Perceived benefits of visiting the MSC; products and services received. RESULTS: MSC visitors (n = 83) and non-visitors (n = 127) did not differ in sociodemographic and injury-related characteristics; 96% of visitors reported learning something new as a result of their visit and 98% would recommend the MSC. During the first 2 years of operation, the MSC made 273 appearances, serving 6086 people. Home child safety products accounted for 71% of the 559 products distributed; educational materials made up 87% of the 7982 services received. Car safety seats accounted for 23% of the products distributed; installations made up 4% of the services received. CONCLUSIONS: This approach to disseminating injury prevention interventions holds promise for enhancing the appeal of safety information and increasing the protection of children.

Effect of bradykinin metabolism inhibitors on evoked hypotension in rats: rank efficacy of enzymes associated with bradykinin-mediated angioedema.

BACKGROUND AND PURPOSE: Inhibition of bradykinin metabolizing enzymes (BMEs) can cause acute angioedema, as demonstrated in a recent clinical trial in patients administered the antihypertensive, omapatrilat. However, the relative contribution of specific BMEs to this effect is unclear and confounded by the lack of a predictive pre-clinical model of angioedema. EXPERIMENTAL APPROACH: Rats were instrumented to record blood pressure and heart rate; inhibitors were infused for 35 min and bradykinin was infused during the last 5 min to elicit hypotension, as a functional marker of circulating bradykinin and relative angioedema risk. KEY RESULTS: In the presence of omapatrilat bradykinin produced dose-dependent hypotension, an effect abolished by B(2) blockade. In the presence of lisinopril (ACE inhibitor), but not candoxatril (NEP inhibitor) or apstatin (APP inhibitor), bradykinin also elicited hypotension. Lisinopril-mediated hypotension was unchanged with concomitant blockade of NEP or NEP/DPPIV (candoxatril+A-899301). However, hypotension was enhanced upon concomitant blockade of APP and further intensified in the presence of NEP inhibition to values not different from omapatrilat alone. CONCLUSIONS AND IMPLICATIONS: We demonstrated that bradykinin is degraded in vivo with an enzyme rank-efficacy of ACE>APP>>NEP or DPPIV. These results suggest the effects of omapatrilat are mediated by inhibition of three BMEs, ACE/APP/NEP. However, dual inhibition of ACE/NEP or ACE/NEP/DPPIV elicits no increased risk of angioedema compared to ACE inhibition alone. Thus, novel BME inhibitors must display no activity against APP to avoid angioedema risk due to high prevalence of ACE inhibitor therapy in patients with diabetes and cardiovascular disease.

Definition of the extended substrate specificity determinants for beta-tryptases I and II.

Tryptases betaI and betaII were heterologously expressed and purified in yeast to functionally characterize the substrate specificity of each enzyme. Three positional scanning combinatorial tetrapeptide substrate libraries were used to determine the primary and extended substrate specificity of the proteases. Both enzymes have a strict primary preference for cleavage after the basic amino acids, lysine and arginine, with only a slight preference for lysine over arginine. betaI and betaII tryptase share similar extended substrate specificity, with preference for proline at P4, preference for arginine or lysine at P3, and P2 showing a slight preference for asparagine. Measurement of kinetic constants with multiple substrates designed for beta-tryptases reveal that selectivity is highly dependent on ground state substrate binding. Coupled with the functional determinants, structural determinants of tryptase substrate specificity were identified. Molecular docking of the preferred substrate sequence to the three-dimensional tetrameric tryptase structure reveals a novel extended substrate binding mode that involves interactions from two adjacent protomers, including P4 Thr-96', P3 Asp-60B' and Glu-217, and P1 Asp-189. Based on the determined substrate information, a mechanism-based tetrapeptide-chloromethylketone inhibitor was designed and shown to be a potent tryptase inhibitor. Finally, the cleavage sites of several physiologically relevant substrates of beta-tryptases show consistency with the specificity data presented here.

Substrate specificity of the human proteasome.

BACKGROUND: Regulated proteolysis by the proteasome is crucial for a broad array of cellular processes, from control of the cell cycle to production of antigens. RESULTS: The rules governing the N-terminal primary and extended substrate specificity of the human 20S proteasome in the presence or absence of 11S proteasome activators (REGalpha/beta and REGgamma) have been elaborated using activity-based proteomic library tools. CONCLUSIONS: The 11S proteasome activators are shown to be important for both increasing the activity of the 20S proteasome and for altering its cleavage pattern and substrate specificity. These data also establish that the extended substrate specificity is an important factor for proteasomal cleavage. The specificities observed have features in common with major histocompatibility complex (MHC) class I ligands and can be used to improve the prediction of MHC class I restricted cytotoxic T-cell responses.

Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries.

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137, 180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors.

Carcinoma of the extrahepatic biliary system: correlation of clinical, pathological, histological and DNA-cytometric parameters with prognosis.

BACKGROUND: Studies concerning the prognosis of all carcinomas of the extrahepatic biliary system together (EBC) do not exist, so far. A detailed and valid histopathological grading-system is lacking and the prognostic relevance of DNA-cytometric parameters is not clear. METHODS: 81 patients with EBC underwent surgical treatment. Clinical, pathological, histomorphological and DNA-cytometric parameters were evaluated. The influence on prognosis was tested in univariate and multivariate analysis. RESULTS: Apart from significant perioperative mortality (17%) survival was markedly dependent on the presence of tumor remnants in resection margins. After incomplete and complete resection, the median survival was 5 and 19 months, respectively (p < 0.0001). Further parameters with significant prognostic relevance were tumor localization, pTNM-stage, period of complaints, presence of gallstones, histological type and DNA-index. Multivariate analysis however demonstrated only two independent prognostic factors: status of resection margins and tumor localization. CONCLUSIONS: Histomorphological- and DNA-cytometric parameters failed to demonstrate an additional significant influence on the prognosis in EBCs. Apart from perioperative mortality, the survival of patients with EBC predominantly depends on tumor localization and the status of resection margins.

Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin.

We have developed a strategy for the synthesis of positional-scanning synthetic combinatorial libraries (PS-SCL) that does not depend on the identity of the P1 substituent. To demonstrate the strategy, we synthesized a tetrapeptide positional library in which the P1 amino acid is held constant as a lysine and the P4-P3-P2 positions are positionally randomized. The 6,859 members of the library were synthesized on solid support with an alkane sulfonamide linker, and then displaced from the solid support by condensation with a fluorogenic 7-amino-4-methylcoumarin-derivatized lysine. This library was used to determine the extended substrate specificities of two trypsin-like enzymes, plasmin and thrombin, which are involved in the blood coagulation pathway. The optimal P4 to P2 substrate specificity for plasmin was P4-Lys/Nle (norleucine)/Val/Ile/Phe, P3-Xaa, and P2-Tyr/Phe/Trp. This cleavage sequence has recently been identified in some of plasmin's physiological substrates. The optimal P4 to P2 extended substrate sequence determined for thrombin was P4-Nle/Leu/Ile/Phe/Val, P3-Xaa, and P2-Pro, a sequence found in many of the physiological substrates of thrombin. Single-substrate kinetic analysis of plasmin and thrombin was used to validate the substrate preferences resulting from the PS-SCL. By three-dimensional structural modeling of the substrates into the active sites of plasmin and thrombin, we identified potential determinants of the defined substrate specificity. This method is amenable to the incorporation of diverse substituents at the P1 position for exploring molecular recognition elements in proteolytic enzymes.

Long-term survival after diagnosis of hepatic metastatic VIPoma: report of two cases with disparate courses and review of therapeutic options.

This report describes two patients with pancreatic cholera caused by vasoactive intestinal polypeptide (VIP)-producing tumors, which originated in the pancreas and showed metastases in both hepatic lobes at time of diagnosis. However, the two tumors displayed remarkably disparate clinical courses. Due to the protracted but progressive course over more than 10 years, a multifaceted therapeutic approach was performed to control symptoms and to improve quality of life. The long-acting somatostatin analog octreotide was the most effective treatment for relieving symptoms and correcting fluid and electrolytes disturbances. The effects of complementary treatments, including systemic chemotherapy and hyperselective chemoembolization, as well as concurrent application of octreotide and prednisolone or interferon with respect to clinical symptoms, VIP levels, and tumor growth are reviewed. Our experience, although small, emphasizes the need for an expert, well-planned, adaptive, and multidisciplinary approach in the care of these complex patients.

Solid support linker strategies.

The selection of an appropriate linker is critical to the success of any strategy for the solid-phase synthesis of small molecule libraries. While the primary function of the linker is to covalently attach the initial substrate to the support, innovative strategies have emerged recently in which linkers fulfill important auxiliary roles. These include the cleavage of compounds into solution leaving no trace of the support attachment site, cleavage via cyclization, cleavage by introduction of additional diversity into the structure, and cleavage whereby portions of the compound are sequentially released into solution.

A reverse-phase HPLC assay for measuring the interaction of polyene macrolide antifungal agents with sterols.

A quick and simple affinity chromatography method for gauging the interaction of polyene antifungal agents with sterols has been developed. The required affinity columns are prepared from a standard C-18 reverse-phase HPLC column by injecting a measured quantity of sterol under conditions where it is completely retained. After the assay, the sterol is eluted with a less polar solvent and the column reused. By comparing the elution volume of a polyene injected onto the sterol-free column (Ve) with that of the polyene injected onto the sterol-doped column (V), an association constant (Ka) for the polyene-sterol complex was determined. Association constants of different amphotericin B-sterol and pimaricin-sterol complexes were determined and correlated with the polyene's ability to induce membrane permeability and its antifungal properties. This procedure provides a new tool for screening polyene macrolides for antifungal therapy.

Evaluation of two rapid methods to screen pathogens from stool specimens.

Two rapid methods, the Enteric Pathogen Screen (EPS) cards of the AutoMicrobic system (Vitek Systems, Inc., Hazelwood, MO) and the Bactigen Salmonella-Shigella latex agglutination (LPA) method (Wampole Laboratories, Cranbury, NJ) were compared with conventional biochemical tests to screen stool cultures having suspicious colonial morphologic characteristics for the presence of Salmonella and Shigella in a study of 481 isolates from stool specimens and 104 stock cultures. Compared with conventional testing, overall, 327 of 394 (83%) clinically irrelevant organisms resulted in a report of no Salmonella spp., Shigella spp., or Yersinia enterocolitica with EPS. Four hundred forty-nine of 457 (98%) of clinically irrelevant organisms yielded negative LPA results. Because the LPA method is faster, eliminates more clinically irrelevant organisms from further testing, and does not require the use of an expensive identification system, the authors believe that it is better suited for direct screening for Salmonella and Shigella for most clinical laboratories.

Rapid antimicrobial susceptibility testing of Gram-negative clinical isolates with the AutoMicrobic system.

Comparisons of the antimicrobial susceptibility patterns of clinical isolates of the family Enterobacteriaceae and Pseudomonas aeruginosa were made by using Kirby-Bauer disk diffusion, the MicroMedia system broth microdilution, and the AutoMicrobic system. The Kirby-Bauer system compared favorably with the MicroMedia system (95% essential correlation with 90% complete agreement). The AutoMicrobic system also compared favorably with both the Kirby-Bauer system (93% essential correlation with 87% complete agreement) and the MicroMedia system (97% essential correlation with 85% complete agreement). These data indicate that the AutoMicrobic system results are comparable to those of the MicroMedia and the Kirby-Bauer systems. Furthermore, the AutoMicrobic system offers a more rapid system (6 to 10 h), than the traditional systems, without sacrificing accuracy (16 to 24 h).