C-terminal processing of yeast Spt7 occurs in the absence of functional SAGA complex

Background  

Spt7 is an integral component of the multi-subunit SAGA complex that is required for the expression of ~10% of yeast genes. Two forms of Spt7 have been identified, the second of which is truncated at its C-terminus and found in the SAGA-like (SLIK) complex.     

Improved high-performance liquid chromatographic determination of guanidino compounds by precolumn dervatization with ninhydrin and fluorescence detection

Ninhydrin has been investigated as a pre-column derivatization reagent for guanidino compounds. The reaction takes place under strongly alkaline conditions, followed by a second step at low pH and elevated temperature. This procedure yields derivatives with favourable fluorescence properties (excitation at 390 nm, emission at 470 nm). Amino acids do not react with ninhydrin under these conditions so that the method can easily be used for biological samples. Reversed-phase HPLC separations of the derivatives of several representative guanidino compounds in human blood have been achieved with gradients consisting of aqueous formic acid and methanol. Fluorescence detection yields quantification limits of about 20 microg L(-1). Hyphenation with electrospray mass spectrometry has been used to confirm the identity of the derivatives.     

Improved capillary electrophoretic separation of nine (fluoro)quinolones with fluorescence detection for biological and environmental samples

A capillary electrophoretic method has been developed for the separation of nine (fluoro)quinolones. Detection is done by fluorescence measurement with broad wavelength band excitation between 240 and 400 nm. Best separation is achieved in a carrier electrolyte containing 50 mM H3PO4 adjusted to pH 7.55-acetonitrile (60:40, v/v). Detection limits are in the low microgl(-1) range. The suitability to real samples has been demonstrated by analyzing blood samples and surface water samples. Sample preconcentration and sample clean-up can easily be done by solid-phase extraction. Different phases based on alkyl- or phenyl-modified silica as well as on polymers have been investigated for this purpose. The method should also be useful for determination of residues of (fluoro)quinolones in food or other matrices.     

Design, synthesis, and study of a mycobactin-artemisinin conjugate that has selective and potent activity against tuberculosis and malaria

Although the antimalarial agent artemisinin itself is not active against tuberculosis, conjugation to a mycobacterial-specific siderophore (microbial iron chelator) analogue induces significant and selective antituberculosis activity, including activity against multi- and extensively drug-resistant strains of Mycobacterium tuberculosis. The conjugate also retains potent antimalarial activity. Physicochemical and whole-cell studies indicated that ferric-to-ferrous reduction of the iron complex of the conjugate initiates the expected bactericidal Fenton-type radical chemistry on the artemisinin component. Thus, this "Trojan horse" approach demonstrates that new pathogen-selective therapeutic agents in which the iron component of the delivery vehicle also participates in triggering the antibiotic activity can be generated. The result is that one appropriate conjugate has potent and selective activity against two of the most deadly diseases in the world.     

Semiquantitative determination of residues of amphetamine in sewage sludge samples

A procedure based on HPLC and mass spectrometric detection has been developed for screening of residues of the illicit drug amphetamine in sewage sludge. Sample pretreatment consisted in extraction by 50 mM formic acid and methanol (80:20 v/v), followed by adjustment of the pH to 10 and preconcentration by SPE at poly(di-vinylbenzene)-N-vinylpyrrolidone. HPLC separation of the extract was done on a C18 RP with a mixture of 50 mM formic acid and methanol (80:20 v/v) as mobile phase. The mass spectrometer was operated in the MS2 and MS3 mode using the transition from m/z 136 to 119 and from m/z 119 to 91. Due to the complex matrix, ionization suppression effects as well as shifts in the sensitivity of the detector within a series of runs could not be fully excluded. Therefore, quantitation was done by standard addition together with external standards, so that semiquantitative results could be obtained down to concentrations of 2 microg/kg sewage sludge. Samples taken from various municipal sewage treatment plants indicate that amphetamine residues are ubiquitous in urban areas.     

A novel technique for on-capillary preconcentration of anionic compounds applied to the trace analysis of rapamycin in human blood by capillary electrophoresis

A capillary electrophoretic (CE) method with UV detection at 278 nm has been developed for analysis of the immunosuppressant rapamycin (sirolimus) in human blood at low microg.L(-1) levels. Separation has been achieved in an acidic carrier electrolyte containing sodium dodecyl sulfate and 20% v/v methanol. For sample cleanup and preconcentration, both an off-line solid-phase extraction step using a silica-based reversed-phase material and a newly developed on-capillary focusing technique have been employed. The subsequent treatment of rapamycin under alkaline conditions leads to a cleavage of the lacton bond of the molecule, generating a negatively charged carboxylic group which allows electrokinetic injection into the CE instrument. During the injection process, the negatively charged analyte migrates into an acidic carrier electrolyte, so that it becomes neutral due to protonation and is focused at the capillary inlet. Injection times of 300 s at -7.5 kV could be applied without band-broadening. Results for real samples indicated that the method is fully suited for routine applications and may be an attractive alternative to established liquid chromatographic techniques.     

Improved liquid chromatographic determination of nine currently used (fluoro)quinolones with fluorescence and mass spectrometric detection for environmental samples

An HPLC method using C18-modified silica as stationary phase has been developed for environmental trace analysis of nine (fluoro)quinolones. Detection is done by fluorescence measurement or MS using the modes of SIM and selected reaction monitoring (SRM). Best separation is achieved with a gradient consisting of 50 mM formic acid and methanol, which is fully compatible with MS coupling. LOQs (S/N of 10) for fluorescence detection are between 10 and 60 microg/L, depending on the analyte. MS detection (SIM and SRM) yields LOQs that are better by a factor of at least an order of magnitude. Sample preconcentration and sample clean-up is accomplished by SPE (preconcentration factor of 1000), leading to LOQs in the low ng/L range. Recoveries of the preconcentration procedure are better than 80% for all analytes. The suitability for real samples has been demonstrated by analyzing surface waters, municipal waste waters, sewage treatment plant effluents, sewage sludge, and sediment taken from rivers and fish ponds. The method should also be useful for determination of residues of (fluoro)quinolones in food or other matrices. The degradation of the (fluoro)quinolones has been examined over 5 days in order to get information about the decomposition rate and the degradation products eventually occurring in the environment.