Characterization of the membranous denitrification enzymes nitrite reductase (cytochrome cd1) and copper-containing nitrous oxide reductase from Thiobacillus denitrificans.

Cytochrome cd1-nitrite reductase and nitrous oxide reductase of Thiobacillus denitrificans were purified and characterized by biochemical and immunochemical methods. In contrast to the generally soluble nature of the denitrification enzymes, these two enzymes were isolated from the membrane fraction of T. denitrificans and remained active after solubilization with Triton X-100. The properties of the membrane-derived enzymes were similar to those of their soluble counterparts from the same organism. Nitrous oxide reductase activity was inhibited by acetylene. Nitrite reductase and nitrous oxide reductase cross-reacted with antisera raised against the soluble enzymes from Pseudomonas stutzeri. The nirS, norBC, and nosZ genes encoding the cytochrome cd1-nitrite reductase, nitric oxide reductase, and nitrous oxide reductase, respectively, from P. stutzeri hybridized with genomic DNA from T. denitrificans. Cross-reactivity and similar N-terminal amino acid and gene sequences suggest that the primary structures of the Thiobacillus enzymes are homologous to the soluble proteins from P. stutzeri.

Pulmonary infiltrations in febrile patients with neutropenia. Risk factors and outcome under empirical antimicrobial therapy in a randomized multicenter study.

BACKGROUND: Different empirical approaches to antimicrobial treatment of lung infiltrates in patients with neutropenia were studied within a prospective, randomized multicenter trial. METHODS: Patients with neutropenia with hematologic malignancies and fever of 38.5 degrees C or higher associated with newly diagnosed lung infiltrates were randomized for an initial therapy with acylaminopenicillin plus aminoglycoside (Group A), third-generation cephalosporin plus aminoglycoside (Group B), or the double beta-lactam combination (Group C), each in combination with rifampin. Nonresponders were given empirical amphotericin B plus 5-fluorocytosine beginning on day 4, day 5, or day 6 under study. RESULTS: Of 295 patients entered, 91.2% were evaluable. Complete response was obtained in 61.3% with no significant difference between treatment groups. The addition of rifampin did not improve treatment results. Only 27.1% of patients achieved a complete response by antibiotic therapy without additional antifungal therapy. Fungi dominated in cases of microbiologically documented infections and were associated with a poorer outcome compared with bacterial pneumonias. The trend of leukocyte counts under study had a highly significant effect on the outcome of infection. CONCLUSIONS: Lung infiltrates in febrile patients with neutropenia represent a high risk of treatment failure. Persistent neutropenia has a significantly adverse effect on the outcome of infection. Incorporation of systemic antifungal agents into first-line therapy, particularly in selected high-risk subgroups, might improve future treatment results. The quality of diagnostic techniques to establish the etiology of pulmonary infiltrates needs to be improved.

[Empirical antimicrobial therapy in neutropenic patients. Results of a multicenter study by the Infections in Hematology Study Group of the Paul Ehrlich Society]. / Empirische antimikrobielle Therapie bei neutropenischen Patienten. Ergebnisse einer multizentrischen Studie der Arbeitsgruppe Infektionen in der Hämatologie der Paul-Ehrlich-Gesellschaft.

BACKGROUND: Severe infections are the predominant cause of treatment failure in patients with high grade malignant hematological disorders undergoing intensive chemotherapy. PATIENTS AND METHODS: In a multicenter trial of the Paul Ehrlich Society (PEG) study group, febrile neutropenic patients with acute leukemias or other high grade hematological malignancies were randomized for a three phase sequential antimicrobial intervention comparing different widely applied regimes for empirical therapy. Patients with clinically documented infections were treated according to a modification depending on the respective source of infection, whereas in patients with microbiologically documented infections, treatment could be adapted to the sesceptibility patterns of detected pathogens. Criteria for evaluation as well as time points for response assessment and treatment escalation were strictly prescribed by the study protocol. RESULTS: Of 1573 evaluable patients, 50.9% had fever of unknown origin (FUO) throughout the study period, 17.1% had lung infiltrates, 14.1% primary bacteremia or fungemia (B/F), 12.6% other clinically documented (CDI) and 5.3% other clinically as well as microbiologically documented infections (CMDI). Cumulative response rate (CR) in patients with FUO was 91.3%, a significant difference between various regimens could not be detected in either of the three treatment phases. Patients with lung infiltrates had a significantly worse treatment outcome as compared to patients with other documented infections or with FUO (61.3% vs 82.9% vs 91.3%). Gram-positive pathogens dominated in case of microbiologically documented infections (MDI), whereas the proportion of fungal infections increased dramatically in MDI with pathogens detected only after more than six days under study. Of numerous prognostic factors analyzed, only the trend in white blood cell counts had a significant impact on treatment outcome. CONCLUSION: Infection-related mortality in neutropenic patients with high grade hematological malignancies can be markedly reduced by a systematically escalating interventional antimicrobial therapy. Early systemic antifungal treatment, especially in patients with lung infiltrates, might further improve treatment results.

Secretory organelles of Paramecium cells (trichocysts) are not remarkably acidic compartments.

Acridine orange (AO) trapping in conjunction with fluorescence microscopy was applied to Paramecium cells. Trichocysts were not labeled when analyzed with an image intensification system (as opposed to a lysosomal population). Only with increasing intensity of ultraviolet light (UV) did trichocysts (and to some extent the cytosol) exhibit orange fluorescence, both effects being paralleled by increasing cell damage. Therefore, in comparison with the reported cytosolic pH (6.8), trichocysts cannot be considered as essentially acidic compartments. This is supported by experiments in vitro, using isolated cortex fragments or isolated fractions of membrane-bounded trichocysts (greater than or equal to 90% non-leaky). Again, during UV illumination orange fluorescence was observed even in the absence of ATP and Mg2+. Furthermore, this AO fluorescence and the condensation state of trichocyst contents were not affected by NH3 or by any of the widely differing ion- and H(+)-exchange inhibitors or ionophores tested. Decondensation of trichocyst contents occurred only when Ca2+ ionophore A23187 or X537A was incorporated into trichocyst membranes and when Ca2+ was then added. In this case all trichocysts partially decondensed within their intact membranes. We conclude that AO might be trapped in trichocysts by the abundant acidic secretory components during observation with UV light, rather than by acidic luminal pH.