Online on-the-spot searching increases use of evidence during consultations in family practice.

OBJECTIVE: The project aimed to search for online evidence in a structured way in consultation with the patient, to investigate whether the evidence discovered changed decisions. METHODS: We developed the "Online on-the-spot" method (OOS) as a part of a quality improvement program. Within a general practice consultation three physicians and two trainees searched in a fixed pattern and sequence the national guidelines of general practitioners developed by the Dutch College of General Practitioners, Clinical Evidence, Trip-database and the British Medical Journal. All GPs who performed this quality improvement program were in favor of the project. RESULTS: During 3 months five GPs registered 365 searches out of 2920 patient-doctor contacts. For each eight patient-doctor contacts there was one online search. Patients were actively involved in 53% of the searches (95%C.I.: 48-57%). On average, two databases were consulted. An answer to the question was found in 87% of cases and in almost half of cases it was relevant new information for the doctor. The GP changed his decision due to the problem in 26% (95%C.I.: 21-29) of cases. At the end of the OOS project, the number of searches within 5 min were significantly higher than at the start: 51% (95% C.I.: 44-59) to 33% (95% C.I.: 24-43), respectively. CONCLUSIONS: The OOS project is a timely answer to the doctors' educational needs in attending to the patient. PRACTICE IMPLICATIONS: OOS could connect the patient, the doctor and the evidence.

Properties of recombinant human plasma procarboxypeptidase U produced in mammalian and insect cells.

BACKGROUND: Carboxypeptidase U (EC 3.4.17.20, TAFIa) is a new member of the metallocarboxypeptidase family circulating in human plasma as a zymogen. It is activated during coagulation and is considered as an important player in the regulation of fibrinolysis. METHODS: Heterologous expression of human plasma procarboxypeptidase U (proCPU, TAFI) was obtained in mammalian cells (C127 and DON) and in insect cells (Sf21 and H5 cells). Conditioned media were purified by cation-exchange chromatography and plasminogen affinity chromatography to yield an essentially pure protein. RESULTS: All systems gave high expression levels (6-20 mg/l). Due to differences in glycosylation of the activation peptide, the recombinant variants of proCPU migrated differently on SDS-PAGE (52-65 kDa). However, after activation, all active recombinant enzymes migrated at 35 kDa, similar to native CPU and no evidence for post-translational modification of the catalytic domains could be detected. For the mammalian cell produced variants, activation was more efficient after desialylation. After activation, CPU showed low solubility (0.2 mg/ml) but was inhibited similarly as native CPU. CONCLUSIONS: Mammalian cell systems were the most efficient for the production of human plasma recombinant proCPU. The obtained zymogen differs with respect to the extent and the heterogeneity of glycosylation but, after activation, the experiments did not reveal any alteration between the recombinant and native protein.

Comparison of the inhibition of human and Trypanosoma cruzi prolyl endopeptidases.

Prolyl endopeptidases (PEPs) have been found in numerous species. Inhibitors of human enzyme could correct cognitive deficits in Alzheimer patients while inhibition of Trypanosoma cruzi PEP could prevent invasion phase in Chagas disease. A structure-activity relationship study carried out in a tetrahydroisoquinoline series allowed to obtain potent competitive inhibitors superior to SUAM-1221. Besides, inhibitors expected to act according to an irreversible mechanism revealed to be superior to JPT-4819, for applications linked to human enzyme inhibition.