Golgi targeting of Drosophila melanogaster beta4GalNAcTB requires a DHHC protein family-related protein as a pilot.

Drosophila melanogaster beta4GalNAcTB mutant flies revealed that this particular N-acetylgalactosaminyltransferase is predominant in the formation of lacdiNAc (GalNAcbeta1,4GlcNAc)-modified glycolipids, but enzymatic activity could not be confirmed for the cloned enzyme. Using a heterologous expression cloning approach, we isolated beta4GalNAcTB together with beta4GalNAcTB pilot (GABPI), a multimembrane-spanning protein related to Asp-His-His-Cys (DHHC) proteins but lacking the DHHC consensus sequence. In the absence of GABPI, inactive beta4GalNAcTB is trapped in the endoplasmic reticulum (ER). Coexpression of beta4GalNAcTB and GABPI generates the active enzyme that is localized together with GABPI in the Golgi. GABPI associates with beta4GalNAcTB and, when expressed with an ER retention signal, holds active beta4GalNAcTB in the ER. Importantly, treatment of isolated membrane vesicles with Triton X-100 disturbs beta4GalNAcTB activity. This phenomenon occurs with multimembrane-spanning glycosyltransferases but is normally not a property of glycosyltransferases with one membrane anchor. In summary, our data provide evidence that GABPI is required for ER export and activity of beta4GalNAcTB.

Capillary electrophoresis-time of flight-mass spectrometry using noncovalently bilayer-coated capillaries for the analysis of amino acids in human urine.

A capillary electrophoresis-time of flight-mass spectrometry (CE-TOF-MS) method for the analysis of amino acids in human urine was developed. Capillaries noncovalently coated with a bilayer of Polybrene (PB) and poly(vinyl sulfonate) (PVS) provided a considerable EOF at low pH, thus facilitating the fast separation of amino acids using a BGE of 1 M formic acid (pH 1.8). The PB-PVS coating proved to be very consistent yielding stable CE-MS patterns of amino acids in urine with favorable migration time repeatability (RSDs <2%). The relatively low sample loading capacity of CE was circumvented by an in-capillary preconcentration step based on pH-mediated stacking allowing 100-nL sample injection (i.e. ca. 4% of capillary volume). As a result, LODs for amino acids were down to 20 nM while achieving satisfactory separation efficiencies. Preliminary validation of the method with urine samples showed good linear responses for the amino acids (R(2) >0.99), and RSDs for peak areas were <10%. Special attention was paid to the influence of matrix effects on the quantification of amino acids. The magnitude of ion suppression by the matrix was similar for different urine samples. The CE-TOF-MS method was used for the analysis of urine samples of patients with urinary tract infection (UTI). Concentrations of a subset of amino acids were determined and compared with concentrations in urine of healthy controls. Furthermore, partial least squares-discriminant analysis (PLS-DA) of the CE-TOF-MS dataset in the 50-450 m/z region showed a distinctive grouping of the UTI samples and the control samples. Examination of score and loadings plot revealed a number of compounds, including phenylalanine, to be responsible for grouping of the samples. Thus, the CE-TOF-MS method shows good potential for the screening of body fluids based on the analysis of endogenous low-molecular weight metabolites such as amino acids and related compounds.

Identification of a new site of sumoylation on Tel (ETV6) uncovers a PIAS-dependent mode of regulating Tel function.

Cell proliferation and differentiation are governed by a finely controlled balance between repression and activation of gene expression. The vertebrate Ets transcriptional repressor Tel (ETV6) and its invertebrate orthologue Yan, play pivotal roles in cell fate determination although the precise mechanisms by which repression of gene expression by these factors is achieved are not clearly defined. Here, we report the identification and characterization of the primary site of sumoylation of Tel, lysine 11 (K11), which is highly conserved in vertebrates (except Danio rerio). We demonstrate that in cells PIAS3 binds to Tel and stimulates sumoylation of K11 in the nucleus. Both Tel monomers and oligomers are efficiently sumoylated on K11 in vitro; but in cells only Tel oligomers are found conjugated with SUMO, whereas sumoylation of Tel monomers is transitory and appears to sensitize them for proteasomal degradation. Mechanistically, sumoylation of K11 inhibits repression of gene expression by full-length Tel. In accordance with this observation, we found that sumoylation impedes Tel association with DNA. By contrast, a Tel isoform lacking K11 (TelM43) is strongly repressive. This isoform results from translation from an alternative initiation codon (M43) that is common to all Tel proteins that also contain the K11 sumoylation consensus site. We find that PIAS3 may have a dual, context-dependent influence on Tel; it mediates Tel sumoylation, but it also augments Tel's repressive function in a sumoylation-independent fashion. Our data support a model that suggests that PIAS-mediated sumoylation of K11 and the emergence of TelM43 in early vertebrates are linked and that this serves to refine spatiotemporal control of gene expression by Tel by establishing a pool of Tel molecules that are available either to be recycled to reinforce repression of gene expression or are degraded in a regulated fashion.

Initial guesses generation for Fluorescence Intensity Distribution Analysis.

The growing number of applications of Fluorescence Intensity Distribution Analysis (FIDA) demands for new approaches in data processing, aiming at increased speed and robustness. Iterative algorithms of parameter estimation, although proven to be universal and accurate, require some initial guesses (IG) of the unknown parameters. An essential component of any data processing technology, IG become especially important in case of FIDA, since even with apparently reasonable, and physically admissible but randomly chosen IG, the iterative procedure may converge to situations where the FIDA model cannot be evaluated correctly. In the present work we introduce an approach for IG generation in FIDA experiments based on the method of moments. IG are generated for the sample parameters: brightness, concentration, and for the parameters related to experimental set-up: background, observation volume profile. A number of analytical simplifications were introduced in order to increase the accuracy and robustness of the numerical algorithms. The performance of the developed method has been tested on number of simulations and experimental data. Iterative fitting with generated IG proved to be more robust and at least five times faster than with an arbitrarily chosen IG. Applicability of the proposed method for quick estimation of brightness and concentrations is discussed.