Design, synthesis, and characterization of a photoactivatable flavocytochrome molecular maquette

We report the construction of a synthetic flavo-heme protein that incorporates two major physiological activities of flavoproteins: light activation of flavin analogous to DNA photolyase and rapid intramolecular electron transfer between the flavin and heme cofactors as in several oxidoreductases. The functional tetra-alpha-helix protein comprises two 62-aa helix-loop-helix subunits. Each subunit contains a single cysteine to which flavin (7-acetyl-10-methylisoalloxazine) is covalently attached and two histidines appropriately positioned for bis-his coordination of heme cofactors. Both flavins and hemes are situated within the hydrophobic core of the protein. Intramolecular electron transfer from flavosemiquinone generated by photoreduction from a sacrificial electron donor in solution was examined between protoporphyrin IX and 1-methyl-2-oxomesoheme XIII. Laser pulse-activated electron transfer from flavin to meso heme occurs on a 100-ns time scale, with a favorable free energy of approximately -100 meV. Electron transfer from flavin to the lower potential protoporphyrin IX, with an unfavorable free energy, can be induced after a lag phase under continuous light illumination. Thus, the supporting peptide matrix provides an excellent framework for the positioning of closely juxtaposed redox groups capable of facilitating intramolecular electron transfer and begins to clarify in a simplified and malleable system the natural engineering of flavoproteins.     

An endo-acting proline-specific oligopeptidase from Treponema denticola ATCC 35405: evidence of hydrolysis of human bioactive peptides

An endo-acting proline-specific oligopeptidase (prolyl oligopeptidase [POPase], EC 3.4.21.26) was purified to homogeneity from the Triton X-100 extracts of cells of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised five successive fast protein liquid chromatography steps. The POPase is a cell-associated 75- to 77-kDa protein with an isoelectric point of ca. 6.5. The enzyme hydrolyzed (optimum pH 6.5) the Pro-pNA bond in carbobenzoxy-Gly-Pro-p-nitroanilide (Z-Gly-Pro-pNA) and bonds at the carboxyl side of proline in several human bioactive peptides, such as bradykinin, substance P, neurotensin, angiotensins, oxytocin, vasopressin, and human endothelin fragment 22-38. The minimum hydrolyzable peptide size was tetrapeptide P3P2P1P'1, while the maximum substrate size was ca. 3 kDa. An imino acid residue in position P1 was absolutely necessary. The hydrolysis of Z-Gly-Pro-pNA was potently inhibited by the following, with the Ki(app) (in micromolar) in parentheses: insulin B-chain (0.7), human endothelin-1 (0.5), neuropeptide Y (1.7), substance P (32.0), T-kinin (4.0), neurotensin (5.0), and bradykinin (16.0). Chemical modification and inhibition studies suggest that the POPase is a serine endopeptidase whose activity depends on the catalytic triad of COOH ... Ser ... His but not on a metal. The amino acid sequence around the putative active-site serine is Gly-Gly-Ser-Asn-Pro-Gly. The enzyme is suggested to contain a reactive cysteinyl residue near the active site. Amino acid residues 4 to 24 of the first 24 N-terminal residues showed a homology of 71% with the POPase precursor from Flavobacterium meningosepticum and considerable homology with the Aeromonas hydrophila POPase. The ready hydrolysis of human bioactive peptides at bonds involving an imino acid residue suggests that enzymes like POPase may contribute to the chronicity of periodontal infections by participating in the peptidolytic processing of those peptides.     

Evaluation of the uncertainty of molecular mass measurement by electrospray ionization mass spectrometry

Electrospray ionization (ES) is a novel method used in mass spectrometry (MS) for producing gas-phase ions from substances in solutions. Common practices for molecular mass estimation from ES spectra summarize the spectrum as a single peak giving no estimate of uncertainty or treat each peak as an independent molecular mass measurement. ES-MS data analysis showed that each peak in an ES spectrum does not always provide an independent measure of molecular mass. Underestimation of measurement uncertainty is a possible result. An elementary time series method, the Yule-Walker equations, was applied to molecular mass estimation from ES data.     

Specific bronchial provocation tests with flour in the diagnosis of occupational bronchial asthma

The gold standard in the diagnosis of occupational asthma is the specific bronchial provocation test (sBPT), but other diagnostic criteria have been proven to have a similar sensitivity, mainly in asthma due to high molecular weight compounds. In order to assess wether some clinical findings can predict the positive response to sBPT, we studied 37 subjects (14 millers and 23 bakers) with suspected occupational asthma who underwent sBPT with wheat flour dust (dust exposure in a small cabin: geometric mean 12.1 mg/m3 for up to 30 min). A positive response to sBPT (FEV1 > 20%) was elicited in 20 subjects (11 early, 4 late, and 5 dual responses). There was no significant difference between subjects with positive or negative sBPT as regards mean age, smoking, length of employment, duration of symptoms, atopy (skin positivity to one or more common allergens) and PD20FEV1 methacholine. The percentage of subjects with work-related symptoms was significantly higher in subjects with positive sBPT with respect to subjects with negative sBPT (81% versus 41.2%, p < 0.01 by chi 2 test); furthermore, FEV1 was significantly lower in subjects with positive sBPT. The percentage of positive skin response to wheat flour extract (mean wheal diameter > or = 3 mm) was mildly but not significantly higher in subjects with positive sBPT (68.4% versus 41.2%). None of the following clinical factors (age < 35 years, asthma symptoms pre-existing occupational exposure, non smokers, atopy and bronchial hyperresponsiveness to methacholine), alone or in combination, were associated with higher prevalence of positive sBPT. We conclude that the response to sBPT in subjects with suspected occupational asthma due to flour dust can not be adequately predicted by other clinical, allergologic and functional data. Therefore, sBPT with flour dust should always be performed in subjects with suspected occupational asthma.     

An expert decision support system for network routing: a case study

The paper presents a case study of the development of an expert decision support system which uses simple heuristic methods for fast determination of routes for simultaneous signals in a transmission network of limited capacity. It illustrates how heuristic solutions can be embodied in a model-based DSS and how the standard decision support literature, although intuitively appealing, provides little practical assistance in system construction or classification