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R A Larsen

Publications and source records attributed to R A Larsen.

At least 19 recordsLinked to original sources
PubMed2006

In vitro-clinical correlations for amphotericin B susceptibility in AIDS-associated cryptococcal meningitis.

Reliable measures of antifungal drug susceptibility are needed. We tested the susceptibility of Cryptococcus neoformans from patients treated with amphotericin B. In vitro susceptibility employed a modified broth macrodilution method. We demonstrate a strong correlation between the quantitative measures of in vitro amphotericin B susceptibility and the quantitative response observed in patients.

AIDS-Related Opportunistic Infections↗
PubMed2001

Cryptococcosis in human immunodeficiency virus-negative patients in the era of effective azole therapy.

We conducted a case study of human immunodeficiency virus (HIV)-negative patients with cryptococcosis at 15 United States medical centers from 1990 through 1996 to understand the demographics, therapeutic approach, and factors associated with poor prognosis in this population. Of 306 patients with cryptococcosis, there were 109 with pulmonary involvement, 157 with central nervous system (CNS) involvement, and 40 with involvement at other sites. Seventy-nine percent had a significant underlying condition. Patients with pulmonary disease were usually treated initially with fluconazole (63%); patients with CNS disease generally received amphotericin B (92%). Fluconazole was administered to approximately two-thirds of patients with CNS disease for consolidation therapy. Therapy was successful for 74% of patients. Significant predictors of mortality in multivariate analysis included age > or =60 years, hematologic malignancy, and organ failure. Overall mortality was 30%, and mortality attributable to cryptococcosis was 12%. Cryptococcosis continues to be an important infection in HIV-negative patients and is associated with substantial overall and cause-specific mortality.

Adolescent↗
PubMed2001

Aspartic peptide hydrolases in Salmonella enterica serovar typhimurium.

Two well-characterized enzymes in Salmonella enterica serovar Typhimurium and Escherichia coli are able to hydrolyze N-terminal aspartyl (Asp) dipeptides: peptidase B, a broad-specificity aminopeptidase, and peptidase E, an Asp-specific dipeptidase. A serovar Typhimurium strain lacking both of these enzymes, however, can still utilize most N-terminal Asp dipeptides as sources of amino acids, and extracts of such a strain contain additional enzymatic activities able to hydrolyze Asp dipeptides. Here we report two such activities from extracts of pepB pepE mutant strains of serovar Typhimurium identified by their ability to hydrolyze Asp-Leu. Although each of these activities hydrolyzes Asp-Leu at a measurable rate, the preferred substrates for both are N-terminal isoAsp peptides. One of the activities is a previously characterized isoAsp dipeptidase from E. coli, the product of the iadA gene. The other is the product of the serovar Typhimurium homolog of E. coli ybiK, a gene of previously unknown function. This gene product is a member of the N-terminal nucleophile structural family of amidohydrolases. Like most other members of this family, the mature enzyme is generated from a precursor protein by proteolytic cleavage and the active enzyme is a heterotetramer. Based on its ability to hydrolyze an N-terminal isoAsp tripeptide as well as isoAsp dipeptides, the enzyme appears to be an isoAsp aminopeptidase, and we propose that the gene encoding it be designated iaaA (isoAsp aminopeptidase). A strain lacking both IadA and IaaA in addition to peptidase B and peptidase E has been constructed. This strain utilizes Asp-Leu as a leucine source, and extracts of this strain contain at least one additional, as-yet-uncharacterized, peptidase able to cleave Asp dipeptides.

Aspartic Acid↗
PubMed2000

Conserved residues Ser(16) and His(20) and their relative positioning are essential for TonB activity, cross-linking of TonB with ExbB, and the ability of TonB to respond to proton motive force.

The cytoplasmic membrane protein TonB couples the proton electrochemical potential of the cytoplasmic membrane to transport events at the outer membrane of Gram-negative bacteria. The amino-terminal signal anchor of TonB and its interaction with the cytoplasmic membrane protein ExbB are essential to this process. The TonB signal anchor is predicted to form an alpha-helix, with a conserved face comprised of residues Ser(16), His(20), Leu(27), and Ser(31). Deletion of either Ser(16) or His(20) or of individual intervening but not flanking residues rendered TonB inactive and unable to assume a proton motive force-dependent conformation. In vivo formaldehyde cross-linking experiments revealed that the ability of this subset of mutants to form a characteristic heterodimer with ExbB was greatly diminished. Replacement of residues 17-19 by three consecutive alanines produced a wild type TonB allele, indicating that the intervening residues (Val, Cys, and Ile) contributed only to spacing. These data indicated that the spatial relationship of Ser(16) to His(20) was essential to function and suggested that the motif HXXXS defines the minimal requirement for the coupling of TonB to the cytoplasmic membrane electrochemical gradient. Deletion of Trp(11) resulted in a TonB that remained active yet was unable to cross-link with ExbB. Because Trp(11) was demonstrably not involved in the actual cross-linking, these results suggest that the TonB/ExbB interaction detected by cross-linking occurred at a step in the energy transduction cycle distinct from the coupling of TonB to the electrochemical gradient.

Bacterial Proteins↗
PubMed2000

Comparison of oral fluconazole and itraconazole for progressive, nonmeningeal coccidioidomycosis. A randomized, double-blind trial. Mycoses Study Group.

BACKGROUND: In previous open-label noncomparative clinical trials, both fluconazole and itraconazole were effective therapy for progressive forms of coccidioidomycosis. OBJECTIVE: To determine whether fluconazole or itraconazole is superior for treatment of nonmeningeal progressive coccidioidal infections. DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: 7 treatment centers in California, Arizona, and Texas. PATIENTS: 198 patients with chronic pulmonary, soft tissue, or skeletal coccidioidal infections. INTERVENTION: Oral fluconazole, 400 mg/d, or itraconazole, 200 mg twice daily. MEASUREMENTS: After 4, 8, and 12 months, a predefined scoring system was used to assess severity of infection. Findings were compared with those at baseline. RESULTS: Overall, 50% of patients (47 of 94) and 63% of patients (61 of 97) responded to 8 months of treatment with fluconazole and itraconazole, respectively (difference, 13 percentage points [95% CI, -2 to 28 percentage points]; P = 0.08). Patients with skeletal infections responded twice as frequently to itraconazole as to fluconazole. By 12 months, 57% of patients had responded to fluconazole and 72% had responded to itraconazole (difference, 15 percentage points [CI, 0.003 to 30 percentage points]; P = 0.05). Soft tissue disease was associated with increased likelihood of response, as in previous studies. Azole drug was detected in serum specimens from all but 3 patients; however, drug concentrations were not helpful in predicting outcome. Relapse rates after discontinuation of therapy did not differ significantly between groups (28% after fluconazole treatment and 18% after itraconazole treatment). Both drugs were well tolerated. CONCLUSIONS: Neither fluconazole nor itraconazole showed statistically superior efficacy in nonmeningeal coccidioidomycosis, although there is a trend toward slightly greater efficacy with itraconazole at the doses studied.

Administration, Oral↗
PubMed2000

Practice guidelines for the management of cryptococcal disease. Infectious Diseases Society of America.

An 8-person subcommittee of the National Institute of Allergy and Infectious Diseases (NIAID) Mycoses Study Group evaluated available data on the treatment of cryptococcal disease. Opinion regarding optimal treatment was based on personal experience and information in the literature. The relative strength of each recommendation was graded according to the type and degree of evidence available to support the recommendation, in keeping with previously published guidelines by the Infectious Diseases Society of America (IDSA). The panel conferred in person (on 2 occasions), by conference call, and through written reviews of each draft of the manuscript. The choice of treatment for disease caused by Cryptococcus neoformans depends on both the anatomic sites of involvement and the host's immune status. For immunocompetent hosts with isolated pulmonary disease, careful observation may be warranted; in the case of symptomatic infection, indicated treatment is fluconazole, 200-400 mg/day for 36 months. For those individuals with non-CNS-isolated cryptococcemia, a positive serum cryptococcal antigen titer >1:8, or urinary tract or cutaneous disease, recommended treatment is oral azole therapy (fluconazole) for 36 months. In each case, careful assessment of the CNS is required to rule out occult meningitis. For those individuals who are unable to tolerate fluconazole, itraconazole (200-400 mg/day for 6-12 months) is an acceptable alternative. For patients with more severe disease, treatment with amphotericin B (0.5-1 mg/kg/d) may be necessary for 6-10 weeks. For otherwise healthy hosts with CNS disease, standard therapy consists of amphotericin B, 0.7-1 mg/kg/d, plus flucytosine, 100 mg/kg/d, for 6-10 weeks. An alternative to this regimen is amphotericin B (0.7-1 mg/kg/d) plus 5-flucytosine (100 mg/kg/d) for 2 weeks, followed by fluconazole (400 mg/day) for a minimum of 10 weeks. Fluconazole "consolidation" therapy may be continued for as along as 6-12 months, depending on the clinical status of the patient. HIV-negative, immunocompromised hosts should be treated in the same fashion as those with CNS disease, regardless of the site of involvement. Cryptococcal disease that develops in patients with HIV infection always warrants therapy. For those patients with HIV who present with isolated pulmonary or urinary tract disease, fluconazole at 200-400 mg/d is indicated. Although the ultimate impact from highly active antiretroviral therapy (HAART) is currently unclear, it is recommended that all HIV-infected individuals continue maintenance therapy for life. Among those individuals who are unable to tolerate fluconazole, itraconazole (200-400 mg/d) is an acceptable alternative. For patients with more severe disease, a combination of fluconazole (400 mg/d) plus flucytosine (100-150 mg/d) may be used for 10 weeks, followed by fluconazole maintenance therapy. Among patients with HIV infection and cryptococcal meningitis, induction therapy with amphotericin B (0.7-1 mg/kg/d) plus flucytosine (100 mg/kg/d for 2 weeks) followed by fluconazole (400 mg/d) for a minimum of 10 weeks is the treatment of choice. After 10 weeks of therapy, the fluconazole dosage may be reduced to 200 mg/d, depending on the patient's clinical status. Fluconazole should be continued for life. An alternative regimen for AIDS-associated cryptococcal meningitis is amphotericin B (0.7-1 mg/kg/d) plus 5-flucytosine (100 mg/kg/d) for 6-10 weeks, followed by fluconazole maintenance therapy. Induction therapy beginning with an azole alone is generally discouraged. Lipid formulations of amphotericin B can be substituted for amphotericin B for patients whose renal function is impaired. Fluconazole (400-800 mg/d) plus flucytosine (100-150 mg/kg/d) for 6 weeks is an alternative to the use of amphotericin B, although toxicity with this regimen is high. In all cases of cryptococcal meningitis, careful attention to the management of intracranial pressure is imperative to assure optimal c

AIDS-Related Opportunistic Infections↗
PubMed1999

Protonmotive force, ExbB and ligand-bound FepA drive conformational changes in TonB.

TonB couples the cytoplasmic membrane protonmotive force (pmf) to active transport across the outer membrane, potentially through a series of conformational changes. Previous studies of a TonB transmembrane domain mutant (TonB-delta V17) and its phenotypical suppressor (ExbB-A39E) suggested that TonB is conformationally sensitive. Here, two new mutations of the conserved TonB transmembrane domain SHLS motif were isolated, TonB-S16L and -H20Y, as were two new suppressors, ExbB-V35E and -V36D. Each suppressor ExbB restored at least partial function to the TonB mutants, although TonB-delta V17, for which both the conserved motif and the register of the predicted transmembrane domain alpha-helix are affected, was the most refractory. As demonstrated previously, TonB can undergo at least one conformational change, provided both ExbB and a functional TonB transmembrane domain are present. Here, we show that this conformational change reflects the ability of TonB to respond to the cytoplasmic membrane proton gradient, and occurs in proportion to the level of TonB activity attained by mutant-suppressor pairs. The phenotype of TonB-delta V17 was more complex than the -S16L and -H20Y mutations, in that, beyond the inability to be energized efficiently, it was also conditionally unstable. This second defect was evident only after suppression by the ExbB mutants, which allow transmembrane domain mutants to be energized, and presented as the rapid turnover of TonB-delta V17. Importantly, this degradation was dependent upon the presence of a TonB-dependent ligand, suggesting that TonB conformation also changes following the energy transduction event. Together, these observations support a dynamic model of energy transduction in which TonB cycles through a set of conformations that differ in potential energy, with a transition to a higher energy state driven by pmf and a transition to a lower energy state accompanying release of stored potential energy to an outer membrane receptor.

Bacterial Outer Membrane Proteins↗
PubMed1999

Early mycological treatment failure in AIDS-associated cryptococcal meningitis.

Cryptococcal meningitis causes significant morbidity and mortality in persons with AIDS. Of 236 AIDS patients treated with amphotericin B plus flucytosine, 29 (12%) died within 2 weeks and 62 (26%) died before 10 weeks. Just 129 (55%) of 236 patients were alive with negative cerebrospinal fluid (CSF) cultures at 10 weeks. Multivariate analyses identified that titer of cryptococcal antigen in CSF, serum albumin level, and CD4 cell count, together with dose of amphotericin B, had the strongest joint association with failure to achieve negative CSF cultures by day 14. Among patients with similar CSF cryptococcal antigen titers, CD4 cell counts, and serum albumin levels, the odds of failure at week 10 for those without negative CSF cultures by day 14 was five times that for those with negative CSF cultures by day 14 (odds ratio, 5.0; 95% confidence interval, 2.2-10.9). Prognosis is dismal for patients with AIDS-related cryptococcal meningitis. Multivariate analyses identified three components that, along with initial treatment, have the strongest joint association with early outcome. Clearly, more effective initial therapy and patient management strategies that address immune function and nutritional status are needed to improve outcomes of this disease.

AIDS-Related Opportunistic Infections↗
PubMed1998

Amphotericin B colloidal dispersion combined with flucytosine with or without fluconazole for treatment of murine cryptococcal meningitis.

Studies with animals and in vitro studies have demonstrated that flucytosine plus amphotericin B or fluconazole has significantly improved mycologic activity against meningitis caused by Cryptococcus neoformans compared to the activity of amphotericin B or fluconazole used alone. However, few doses have been tested in combination. This study evaluated the antifungal efficacy of amphotericin B colloidal dispersion (ABCD) combined with flucytosine with and without fluconazole in a murine model of cryptococcal meningitis. The following dosages were tested: ABCD at 0 to 12.5 mg/kg of body weight given intravenously 3 days/week, flucytosine at 0 to 110 mg/kg/day, and fluconazole at 0 to 50 mg/kg/day. Meningitis was established in male BALB/c mice by intracerebral injection of C. neoformans. Treatment with flucytosine with or without fluconazole dissolved in the sole source of drinking water was started on day 2; animals were sacrificed at 16 days, and the numbers of fungal colonies in the brain were quantified. A survival rate of 100% was achieved with ABCD plus flucytosine without fluconazole; however, the addition of fluconazole was required to prevent weight loss (P < 0.00001) and to achieve the maximum antifungal effect (P < 0.00001). The only region of dose combinations for which the 99% confidence intervals were less than 100 CFU/g of brain was defined by ABCD at 5.0 to 7.5 mg/kg combined with flucytosine at 20 to 60 mg/kg/day and fluconazole at 30 to 40 mg/kg/day. The triple combination of ABCD plus flucytosine and fluconazole was necessary to achieve the greatest antifungal activity.

Amphotericin B↗
PubMed1997

Treatment of histoplasmosis with fluconazole in patients with acquired immunodeficiency syndrome. National Institute of Allergy and Infectious Diseases Acquired Immunodeficiency Syndrome Clinical Trials Group and Mycoses Study Group.

PURPOSE: This study assesses the efficacy and safety of fluconazole therapy in patients with acquired immunodeficiency syndrome (AIDS) and mild to moderately severe manifestations of disseminated histoplasmosis. PATIENTS AND METHODS: This was a multicenter, open-label, nonrandomized prospective trial. All patients had AIDS and disseminated histoplasmosis. Patients were treated with 1,200 mg of fluconazole given by mouth once on the first day, then 600 mg once daily for 8 weeks, and those patients who improved clinically were then assigned fluconazole maintenance therapy 200 mg once daily for at least 1 year. Interim analysis revealed a high failure rate (10 of 20, 50%), causing revision of the protocol to increase the fluconazole dose to 1,600 mg given once on the first day, then 800 mg once daily, and the duration to 12 weeks for induction therapy and then 400 mg daily for 1 year for maintenance therapy. MEASUREMENTS AND MAIN RESULTS: Thirty-six of 49 patients (74%; 95% confidence interval [CI]: 59% to 85%) with mild to moderately severe clinical manifestations who entered into the revised study responded to 800 mg of fluconazole daily for 12 weeks as induction therapy. Of the seven patients who failed induction therapy because of progression of histoplasmosis, one died of the infection. Of 36 patients who entered into the maintenance phase of the study receiving 400 mg of fluconazole daily for 1 year, 11 (30.5%) relapsed, including one who died (2.8%). Two of the 49 patients (4.1%) were removed because of grade 4 adverse events, alkaline phosphatase elevation for one and aspartate aminotransferase elevation in the other. The relapse-free rate at 1 year was 53% (95% CI: 32% to 89%), prompting closure of the study. CONCLUSIONS: Fluconazole 800 mg daily is a safe and moderately effective induction therapy for mild or moderately severe disseminated histoplasmosis in patients with AIDS. On the basis of historic comparison, fluconazole 400 mg daily is less effective than itraconazole 200 to 400 mg daily or amphotericin B 50 mg given weekly as maintenance therapy to prevent relapse.

AIDS-Related Opportunistic Infections↗
PubMed1997

Effect of severity of meningitis on fungicidal activity of flucytosine combined with fluconazole in a murine model of cryptococcal meningitis.

We studied the effect of the severity of meningitis on the response to therapy with fluconazole and flucytosine in a murine model of cryptococcal meningitis. Meningitis was established by intracerebral injection of Cryptococcus neoformans. The severity of meningitis was varied by delaying the onset of treatment from 3 to 7 days. Animals were sacrificed after 14 days of treatment, and the numbers of C. neoformans per gram of brain tissue were quantified. The range of effective dose combinations of fluconazole and flucytosine became progressively reduced as the severity of meningitis increased. The magnitude of treatment effect, as measured by the numbers of CFU/gram of brain tissue, was also reduced with increasing severity of meningitis. In this model, as the severity of meningitis increases, higher doses of fluconazole are required to achieve equivalent levels of activity. The combination of fluconazole and flucytosine appears to have the most-potent antifungal effects. This is most readily observed in animals with more-severe meningitis.

Animals↗
PubMed1997

Regions of Escherichia coli TonB and FepA proteins essential for in vivo physical interactions.

The transport of Fe(III)-siderophore complexes and vitamin B12 across the outer membrane of Escherichia coli is an active transport process requiring a cognate outer membrane receptor, cytoplasmic membrane-derived proton motive force, and an energy-transducing protein anchored in the cytoplasmic membrane, TonB. This process requires direct physical contact between the outer membrane receptor and TonB. Previous studies have identified an amino-terminally located region (termed the TonB box) conserved in all known TonB-dependent outer membrane receptors as being essential for productive energy transduction. In the present study, a mutation in the TonB box of the ferric enterochelin receptor FepA resulted in the loss of detectable in vivo chemical cross-linking between FepA and TonB. Protease susceptibility studies indicated this effect was due to an alteration of conformation rather than the direct disruption of a specific site of physical contact. This suggested that TonB residue 160, implicated in previous studies as a site of allele-specific suppression of TonB box mutants, also made a conformational rather than a direct contribution to the physical interaction between TonB and the outer membrane receptors. This possibility was supported by the finding that TonB carboxyl-terminal truncations that retained Gln-160 were unable to participate in TonB-FepA complex formation, indicating that this site alone was not sufficient to support the physical interactions involved in energy transduction. These studies indicated that the final 48 residues of TonB were essential to this physical interaction. This region contains a putative amphipathic helix which could facilitate TonB-outer membrane interaction. Amino acid replacements at one site in this region were found to affect energy transduction but did not appear to greatly alter TonB conformation or the formation of a TonB-FepA complex. The effects of amino acid substitutions at several other TonB sites were also examined.

Bacterial Outer Membrane Proteins↗
PubMed1996

Identification of patients with acute AIDS-associated cryptococcal meningitis who can be effectively treated with fluconazole: the role of antifungal susceptibility testing.

No method currently exists to predict which patients with acute AIDS-associated cryptococcal meningitis can be effectively treated with fluconazole. The objective of this study was to determine the relationship of cryptococcal susceptibility to fluconazole, along with clinical variables, to the risk of treatment failure for patients with acute AIDS-associated cryptococcal meningitis. Results of in vitro fluconazole susceptibility testing of cryptococcal isolates and data from two clinical trials were analyzed. Susceptibility to fluconazole was determined by means of both microtiter and macrobroth (M27-P) dilution methods. Treatment was defined as successful if the patient was alive at 10 weeks and if a cerebrospinal fluid culture was sterile at that time. Seventy-six patients receiving fluconazole +/- flucytosine were included; therapy failed for 19. Patients whose therapy failed were more likely to have a positive blood and urine culture and a higher titer in serum and cerebrospinal fluid of cryptococcal antigen, and the MIC of fluconazole against their isolates (as determined by the microtiter method) was more likely to be higher; they were less likely to have received flucytosine. Logistic regression modeling revealed that a negative blood culture, a low MIC of fluconazole (per the microtiter method), and treatment with flucytosine were factors independently associated with successful treatment.

AIDS-Related Opportunistic Infections↗
PubMed1996

Structure/activity studies of anti-inflammatory peptides based on a conserved peptide region of the lectin domain of E-, L- and P-selectin.

Previously, it was established that the peptide YYWIGIRK-NH2 inhibits both myeloid cell adhesion to selectins in vitro and neutrophil influx into inflammatory sites in vivo (Briggs et al., 1995). Initial structure/activity studies revealed that at least one Y residue at the N-terminus of the peptide was essential for these bioactivities but that the C-terminal K residue was unnecessary for inhibitory activity. We have now synthesized a new series of peptides which contain single residue substitutions at each position of the reference peptide, YYWIGIR-NH2, and have tested these peptides for inhibitory activity in a selectin cell binding assay. In addition, peptides containing single D-amino acids at selected positions, or an all D-configured reference peptide sequence, or the retro-inverso version (rigiwyy-NH2) of the reference peptide sequence have also been analyzed for inhibitory activity in the same assays. Finally, the ability of the reference peptide and a specifically designed control sequence (YY(AIB)IGIR-NH2) to discriminate between potential synthetic saccharide ligands, including sialyl-Lewis x, Lewis x, and sialyl-N-acetyl-lactosamine, was investigated using isothermal titration calorimetry. The results of these studies demonstrate that whereas many single amino acid substitutions are tolerated in the peptide without complete loss of inhibitory activity, substitution at some positions (e.g., the W residue) results in relatively inactive compounds, clearly pointing to the importance of these residues in making critical contacts with the appropriate saccharide ligand. Titration calorimetry revealed that the reference peptide does not discriminate between Lewis x or sialyl-Lewis x in vitro, but binds these saccharides with nearly 40-fold higher affinity (KD 25 microM) than the nonfucosylated trisaccharide, sialyl-N-acetyl-lactosamine. We can infer from these studies that the presence of a sialyl group per se, is not a requisite for complex formation between the reference peptide and its saccharide ligand. Substitution of single D-amino acid residues at various positions in the reference peptide sequence reduces or eliminates all inhibitory properties. However, the all D-configured peptide or the retro-inverso peptide sequence have greater activity than the all L-configured reference peptide in the in vitro biological assays, and each was an effective inhibitor of neutrophil infiltration in a thioglycolate-induced mouse peritonitis model. These results, combined with the results of titration, allow us to conclude that binding between the reference peptide and its saccharide ligand, which affords its inhibitory properties, is mediated by the presence of a contiguous, nonpolar surface, or face, presented at the N-terminus of the reference peptide, likely encompassing the sequence YYWI. Furthermore, the W plays a critical role in binding, probably through formation of an essential hydrogen bond with a suitably juxtaposed group carried on the saccharide ligand.

Amino Acid Sequence↗
PubMed1996

Effect of fluconazole on fungicidal activity of flucytosine in murine cryptococcal meningitis.

Both animal and in vitro studies have demonstrated that combinations of flucytosine with amphotericin B and with fluconazole have significantly improved activity against cryptococcal meningitis compared with the activity of each drug used alone. However, very few dose levels of these agents have been tested in combination. This study evaluated the efficacy of fluconazole plus flucytosine in a murine model of cryptococcal meningitis over a broad range of dose combinations (fluconazole, 0 to 40 micrograms/g of body weight per day; flucytosine, 0 to 200 micrograms/g/day). Both drugs were dissolved in drinking water, with treatment on days 2 to 11. In this highly reproducible model, fluconazole had a dramatic effect on the fungicidal activity of flucytosine. Flucytosine at dose levels of as much as 200 micrograms/g/day alone or in combination with low doses of fluconazole had minimal fungicidal activity, whereas in combination with fluconazole at 24 to 40 micrograms/g/day, flucytosine showed fungicidal activity in the range of 45 to 65% of the animals treated at doses of 40 to 100 micrograms/g/day. This striking effect of fluconazole is consistent with the results of both in vitro and clinical studies. In the clinic, the use of flucytosine is often limited by severe toxicity, while toxicity is rarely observed with fluconazole. These results suggest that when flucytosine is given with higher doses of fluconazole, the maximum therapeutic effect of the former in the clinic may be observed at dose levels that are far less than the doses commonly employed (150 micrograms/g daily).

Animals↗
PubMed1996

Identification of TonB homologs in the family Enterobacteriaceae and evidence for conservation of TonB-dependent energy transduction complexes.

The transport of Fe(III)-siderophore complexes and vitamin B12 across the outer membrane of Escherichia coli requires the TonB-dependent energy transduction system. A set of murine monoclonal antibodies (MAbs) was generated against an E. coli TrpC-TonB fusion protein to facilitate structure and function studies. In the present study, the epitopes recognized by these MAbs were mapped, and their distribution in gram-negative organisms was examined. Cross-species reactivity patterns obtained against TonB homologs of known sequence were used to refine epitope mapping, with some epitopes ultimately confirmed by inhibition experiments using synthetic polypeptides. Epitopes recognized by this set of MAbs were conserved in TonB homologs for 9 of 12 species in the family Enterobacteriaceae (including E. coli), including previously unidentified TonB homologs in Shigella, Citrobacter, Proteus, and Kluyvera species. These homologs were also detected by a polyclonal alpha-TrpC-TonB serum that additionally recognized the known Yersinia enterocolitica TonB homolog and a putative TonB homolog in Edwardsiella tarda. These antibody preparations failed to detect the known TonB homologs of either Pseudomonas putida or Haemophilus influenzae but did identify potential TonB homologs in several other nonenteric gram-negative species. In vivo chemical cross-linking experiments demonstrated that in addition to TonB, auxiliary components of the TonB-dependent energy transduction system are broadly conserved in members of the family Enterobacteriaceae, suggesting that the TonB system represents a common system for high-affinity active transport across the gram-negative outer membrane.

Amino Acid Sequence↗