trans-2-phenylcyclopropylamine is a substrate for and inactivator of horseradish peroxidase

Horseradish peroxidase (HRP) is well known for mediating the electron-transfer oxidation of electron-rich aromatic 'donors' such as phenols and anilines, but has not been described to oxidize aliphatic amines. We here confirm the inability of HRP to oxidize typical aliphatic amines, even those which would exist significantly as free bases at the operative pH. In contrast, trans-2-phenylcyclopropylamine (2-PCPA) is both a substrate (turnover product is cinnamaldehyde) and a time-dependent inactivator of HRP. These activities of 2-PCPA are consistent with either a concerted or rapid sequential one-electron-oxidation/ring-opening to give an intermediate capable of covalent binding to the enzyme. 2-PCPA is the first known example of a simple aliphatic amine which serves as a substrate for HRP under turnover conditions.     

Agricultural safety efforts by county health departments in Wisconsin

OBJECTIVE: The authors sought to improve the agriculture safety prevention efforts of county health departments in Wisconsin by examining current programs, staffs' perceptions of the farm safety problem, and the need for new resources. METHODS: A survey instrument was completed by a professional staff member of the local health department in each of Wisconsin's 69 counties. RESULTS: Usable responses were obtained from 84% of the counties. Forty-five percent of the responding staff members conducted some agricultural safety and health programs, most often health screenings or group meetings conducted collaboratively with county agricultural Extension agents. There were no major differences in county demographics or other service provision variables between staff members who conducted programs and those who did not. Staff members perceived the largest barriers to better safety as lack of staff time and difficulty getting farmers to attend safety programs. Most failed to place more emphasis on training agricultural workers to permanently correct hazards than on training them to work safely around hazards. However, the staff members ranked safety inspection checklists as the most needed new material and ranked Extension agents and farmers as the most appropriate people to conduct inspections using such checklists. CONCLUSION: County public health professionals want more staff time and new materials to increase the effectiveness of their agricultural safety efforts. Encouraging agricultural workers and family members to identify and correct hazards would be a more effective use of staff time than training people to work safely around hazards.     

Maintaining excellence: deliberate practice and elite performance in young and older pianists

Two studies investigated the role of deliberate practice in the maintenance of cognitive-motor skills in expert and accomplished amateur pianists. Older expert and amateur pianists showed the normal pattern of large age-related reductions in standard measures of general processing speed. Performance on music-related tasks showed similar age-graded decline for amateur pianists but not for expert pianists, whose average performance level was only slightly below that of young expert pianists. The degree of maintenance of relevant pianistic skills for older expert pianists was predicted by the amount of deliberate practice during later adulthood. The role of deliberate practice in the active maintenance of superior domain-specific performance in spite of general age-related decline is discussed.     

A frizzled homolog functions in a vertebrate Wnt signaling pathway

BACKGROUND: Wnts are secreted proteins implicated in cell-cell interactions during embryogenesis and tumorigenesis, but receptors involved in transducing Wnt signals have not yet been definitively identified. Members of a large family of putative transmembrane receptors homologous to the frizzled protein in Drosophila have been identified recently in both vertebrates and invertebrates, raising the question of whether they are involved in transducing signals for any known signaling factors. RESULTS: To test the potential involvement of frizzled homologs in Wnt signaling, we examined the effects of overexpressing rat frizzled-1 (Rfz-1) on the subcellular distribution of Wnts and of dishevelled, a cytoplasmic component of the Wnt signalling pathway. We demonstrate that ectopic expression of Rfz-1 recruits the dishevelled proten-as well as Xenopus Wnt-8 (Xwnt-8), but not the functionally distinct Xwnt-5A-to the plasma membrane. Moreover, Rfz-1 is sufficient to induce the expression of two Xwnt-8-responsive genes, siamois and Xnr-3, in Xenopus explants in a manner which is antagonized by glycogen synthase kinase-3, which also antagonizes Wnt signaling. When Rfz-1 and Xwnt-8 are expressed together in this assay, we observe greater induction of these genes, indicating that Rfz-1 can synergize with a Wnt. CONCLUSIONS: The results demonstrate that a vertebrate frizzled homolog is involved in Wnt signaling in a manner which discriminates between functionally distinct Wnts, which involves translocation of the dishevelled protein to the plasma membrane, and which works in a synergistic manner with Wnts to induce gene expression. These data support the likely function of frizzled homologs as Wnt receptors, or as components of a receptor complex.     

Inhibition of TEM-2 beta-lactamase from Escherichia coli by clavulanic acid: observation of intermediates by electrospray ionization mass spectrometry

Clavulanic acid, the therapeutically important inhibitor of beta-lactamases containing a nucleophilic serine residue at their active sites, inhibits Escherichia coli TEM-2 beta-lactamase via a complex mechanism. Electrospray ionization mass spectrometry (ESIMS) studies revealed that a minimum of four different modified proteins are formed upon incubation of clavulanate with the TEM-2 enzyme. These exhibit mass increments relative to the unmodified TEM-2 beta-lactamase of 52, 70, 88, and 155 Da. Time course studies implied that no long-lived forms of clavulanate-inhibited TEM-2 beta-lactamase retain the carbons of the oxazolidine ring of clavulanate. The absence of a 199 Da increment to unmodified TEM-2 suggests rapid decarboxylation of clavulanate upon binding to the enzyme. Proteolytic digestions of purified forms of clavulanate inhibited TEM-2 beta-lactamase followed by analyses using high-performance liquid chromatography coupled to ESIMS (HPLC-ESIMS) and chemical sequencing were used to provide positional information on the modifications to the enzyme. Increments of 70 and 80 Da increments were shown to be located in a peptide containing Ser-70. A further 70 Da mass increment, assigned as a beta-linked acrylate, was localized to a peptide containing Ser-130. A mechanistic scheme for the reaction of clavulanate with TEM-2 beta-lactamase is proposed in which acylation at Ser-70 and subsequent decarboxylation is followed either by cross-linking with Ser-130 to form a vinyl ether or by reformation of unmodified enzyme via a Ser-70 linked (hydrated) aldehyde. Purified cross-linked vinyl ether was observed to slowly convert under acidic conditions to a Ser-70 linked (hydrated) aldehyde with concomitant conversion of Ser-130 to a dehydroalanyl residue.