Mass spectrometric quantitation of covalently bound cell wall proteins in Saccharomyces cerevisiae

The cell wall of yeast consists of an internal skeletal layer and an external layer of glycoproteins covalently linked to the stress-bearing polysaccharides. The cell wall protein (CWP) population consists of over 20 different proteins, and may vary in composition. We present two complementary methods for quantifying CWPs, based on isobaric tagging and tandem MS: (1) absolute quantitation of individual CWPs, allowing estimation of surface densities; and (2) relative quantitation of CWPs, allowing monitoring of the dynamics of the CWP population. For absolute quantitation, we selected a representative group of five proteins (Cwp1p, Crh1p, Scw4p, Gas1p, and Ecm33p), which had 67 × 103, 44 × 103, 38 × 103, 11 × 103 and 6.5 × 103 of wall-bound copies per cell, respectively. As Cwp1p is predominantly incorporated in the birth scar, this corresponds to a protein density of c. 22 × 103 copies μm−2. For relative quantitation, we compared wild-type cells to gas1Δ cells, in which the cell wall integrity pathway is constitutively activated. The levels of Crh1p, Crh2p, Ecm33p, Gas5p, Pst1p and Pir3p increased about three- to fivefold, whereas the level of Scw4p was significantly decreased. We propose that our methods are widely applicable to other fungi

Impairment of mitochondrial protein synthesis, diogenetic and bioenergetic aspects

In vrijwel alle dierlijke en plantaardige cellen komen mitochondriën voor. Mitochondriën zijn structuren die omgeven zijn door een dubbele membraan en die de energievoorziening van de cel verzorgen. Een cel kan het dan ook niet zonder goed functionerende mitochondriën stellen. Mitochondriën kunnen zelf een aantal eiwitten maken. Deze ei witsynthese wordt specifiek geremd door een aantal stoffen die ook de eiwitsynthese in bacteriën remmen, maar die de eiwitsynthese in de cel zelf ongemoeid laten. Dergelijke stoffen zijn bijvoorbeeld tetracycline, chlooramfenicol en thiamfenicol. Al deze antibiotica vinden klinische toepassing ter bestrijding of preventie van infecties. In Hoofdstuk II is beschreven in hoeverre behandeling van ratten met thiamfenicol invloed heeft op het functioneren van rnitochondriën. Deze onderzoekingen zijn gedaan in het weefsel dat de darmwand bekleedt, het darmepitheel, omdat juist dit weefsel voortdurend en met hoge snelheid wordt vernieuwd en dus een actieve eiwitsynthese heeft. ... Zie: Samenvatting

Quantitation of newly synthesized proteins by pulse labeling with azidohomoalanine

Measuring protein synthesis and degradation rates on a proteomic scale is an important step toward modeling the kinetics in complicated cellular response networks. A gel-free method, able to quantify changes in the formation of new proteins on a 15 min timescale, compatible with mass spectrometry is described. The methionine analogue, azidohomoalanine (azhal), is used to label newly formed proteins during a short pulse-labeling period following an environmental switch in Escherichia coli. Following digestion a selective reaction against azhal-containing peptides is applied to enrich these peptides by diagonal chromatography. This technique enables quantitation of hundreds of newly synthesized proteins and provides insight into immediate changes in newly synthesized proteins on a proteomic scale after an environmental perturbatio

Nuclear Domains Enriched in RNA 3′-processing Factors Associate with Coiled Bodies and Histone Genes in a Cell Cycle–dependent Manner

Nuclear domains, called cleavage bodies, are enriched in the RNA 3′-processing factors CstF 64 kDa and and CPSF 100 kDa. Cleavage bodies have been found either overlapping with or adjacent to coiled bodies. To determine whether the spatial relationship between cleavage bodies and coiled bodies was influenced by the cell cycle, we performed cell synchronization studies. We found that in G1 phase cleavage bodies and coiled bodies were predominantly coincident, whereas in S phase they were mostly adjacent to each other. In G2 cleavage bodies were often less defined or absent, suggesting that they disassemble at this point in the cell cycle. A small number of genetic loci have been reported to be juxtaposed to coiled bodies, including the genes for U1 and U2 small nuclear RNA as well as the two major histone gene clusters. Here we show that cleavage bodies do not overlap with small nuclear RNA genes but do colocalize with the histone genes next to coiled bodies. These findings demonstrate that the association of cleavage bodies and coiled bodies is both dynamic and tightly regulated and suggest that the interaction between these nuclear neighbors is related to the cell cycle–dependent expression of histone genes

Open and Laparoscopic Colposuspension in Girls with Refractory Urinary Incontinence

IntroductionLower urinary tract symptoms (LUTS) are very common in children. Standard treatments consist of urotherapy, antibiotic prophylaxis, anti-muscarinics, physical therapy, and the treatment of coexisting constipation. A small group of girls also present with stress incontinence or with stress-induced urge incontinence. In cases of persistent LUTS due to congenital bladder neck insufficiency (BNI), surgical treatment might be considered. The aim of this paper is to assess the results of open and laparoscopic colposuspension in children with refractory urinary incontinence (UI).Materials and methodsThe results of 18 open and 18 laparoscopic consecutive colposuspensions were analyzed. All patients had UI and failed conservative treatment. BNI was proven by repeated perineal ultrasound and video-urodynamic study. The laparoscopic procedure was performed preperitoneally and the open procedure was via a transverse lower abdominal incision. The same postoperative protocol was used in both groups.ResultsThe mean operation time was 65 min for the open and 90 min for the lap procedure (p < 0.05). Full success was achieved in 7/18 in the open and in 8/18 in the lap group and partial response was seen in 3/18 and in 5/18, respectively (p = 0.64). No intraoperative complications occurred in this cohort.ConclusionOpen and laparoscopic colposuspension can be used to treat refractory UI in children with BNI when non-invasive methods fail