Screening for Transglutaminase-Catalyzed Modifications by Peptide Mass Finger Printing Using Multipoint Recalibration on Recognized Peaks for High Mass Accuracy

Detection of posttranslational modifications is expected to be one of the major future experimental challenges for proteomics. We describe herein a mass spectrometric procedure to screen for protein modifications by peptide mass fingerprinting that is based on post-data acquisition improvement of the mass accuracy by exporting the peptide mass values into analytical software for multipoint recalibration on recognized peaks. Subsequently, the calibrated peak mass data set is used in searching for modified peptides, i.e., peptides possessing specific mass deviations. In order to identify the location of Lys- and Gln-residues available for transglutaminase-catalyzed isopeptide bond formation, mammalian small heat shock proteins (sHsps) were screened for labeling with the two hexapeptide probes GQDPVR and GNDPVK in presence of transglutaminase. Peptide modification due to cross-linking of the GQDPVR hexa-peptide probe was detected for C-terminal Lys residues. Novel transglutaminase-susceptible Gln sites were identified in two sHsps (Q31/Q27 in Hsp20 and HspB2, respectively), by cross-linking of the GNDPVK hexapeptide probe. Deamidation of specific Gln residues was also detected, as well an isopeptide derived from intramolecular Gln-Lys isopeptide bond formation. We conclude that peptide mass fingerprinting can be an efficient way of screening for various posttranslational modifications. Basically any instrumentation for MALDI mass spectrometry can be used, provided that post-data acquisition recalibration is applied

Physical activity, Sedentary behaviour, use of electronic media, and snacking among youth: An international study

This study examined physical activity, sedentary behaviours, location of electronic media and snacking among children from five countries. These variables were assessed by ecological momentary assessment (EMA) using a free-time diary. Data were obtained from 812 secondary-school students (348 male, 464 female) aged from 12 to 18 years in United Kingdom, China, Hungary, Romania and Slovakia. We found that less than half the students met the recommended guideline of 60 minutes daily physical activity (48% of British, 40% of Romanian, 34% of Slovakian, 20% of Hungarian and only 4% of Chinese students met this criterion). Ninety-six percent of British and 86% of Hungarian youth had more than one TV set in their home, followed by Romanian (64%), Slovakian (64%) and Chinese (29%) counterparts. Most British (73%) youths had televisions in their bedroom, followed by Hungarians (66%), Romanians (37%), Slovakians (35%) and Chinese (4%). When compared to females, male students spent significantly more time on TV/DVD/ video viewing (on average 110.7 vs 90.2 minutes/day; p<.001) and playing computer games (on average 34.0 vs 10.5 minutes/day; p<.001). Students who had a TV in their bedroom spent more time watching TV compared to those without a TV in their bedroom (on average 109 vs 91 minutes/day, p<.001). Higher levels of TV viewing were associated with more snack food consumption (r=.13, p<.01). In order to promote less TV viewing and snacking, it may be useful to keep TVs out of the bedrooms of children and adolescents

Prevalence of sedentary behaviour in young people in Romania and Slovakia

Sedentary behaviour is becoming a popular area of health research, but most studies report data on samples from Australia, the UK and USA, and on a narrow range of behaviours. The present study reports on the prevalence of multiple sedentary behaviours in a sample of secondary school students (n = 635; mean age 16.0 years) from Romania and Slovakia. Ecological Momentary Assessment diaries were used to record multiple behaviours across weekdays and weekend days. Results showed high levels of many sedentary behaviours, particularly for screen time at weekends. Other behaviours included homework, sedentary socializing and motorized transport. Gender, age and country differences were evident for some behaviours. Interventions may need to account for socio-demographic moderators, and studies need to assess multiple sedentary behaviours. © North West Counties Physical Education Association, SAGE Publications 2012

TLR9 regulates the mycobacteria-elicited pulmonary granulomatous immune response in mice through DC-derived Notch ligand delta-like 4

TLR9 activation is important for the maintenance of mycobacteria-elicited pulmonary granulomatous responses, hallmarks of protective immune responses following mycobacterial infection. However, the mechanism or mechanisms underlying this effect of TLR9 are not clear. Here, we show that Tlr9-deficient mice challenged with a Mycobacterium antigen display an altered Th17 cytokine profile, decreased accumulation of granuloma-associated myeloid DCs, and profoundly impaired delta-like 4 (dll4) Notch ligand expression. Mechanistic analysis revealed that WT bone marrow–derived DCs but not macrophages promoted the differentiation of Th17 cells from bacillus Calmette-Guérin–challenged (BCG-challenged) lung CD4+ T cells. Both lung and bone marrow DCs isolated from Tlr9-deficient mice inoculated with Mycobacterium antigen expressed lower levels of dll4 Notch ligand than the same cells isolated from WT mice. Passively immunizing WT mice with neutralizing antibodies specific for dll4 during granuloma formation resulted in larger granulomas and lower levels of Th17-related cytokines. In addition, dll4 specifically regulated Th17 activation in vitro. Together, these results suggest dll4 plays an important role in promoting Th17 effector activity during a mycobacterial challenge. Furthermore, TLR9 seems to be required for optimal dll4 expression and the regulation of Mycobacterium antigen–elicited granuloma formation in mice

The small heat-shock proteins HSPB2 and HSPB3 form well-defined heterooligomers in a unique 3 to 1 subunit ratio

Contains fulltext : 75686.pdf (Publisher’s version ) (Open Access

Tissue transglutaminase inhibits the TRPV5-dependent calcium transport in an N-glycosylation-dependent manner.

Contains fulltext : 109998.pdf (publisher's version ) (Closed access)Tissue transglutaminase (tTG) is a multifunctional Ca(2+)-dependent enzyme, catalyzing protein crosslinking. The transient receptor potential vanilloid (TRPV) family of cation channels was recently shown to contribute to the regulation of TG activities in keratinocytes and hence skin barrier formation. In kidney, where active transcellular Ca(2+) transport via TRPV5 predominates, the potential effect of tTG remains unknown. A multitude of factors regulate TRPV5, many secreted into the pro-urine and acting from the extracellular side. We detected tTG in mouse urine and in the apical medium of polarized cultures of rabbit connecting tubule and cortical collecting duct (CNT/CCD) cells. Extracellular application of tTG significantly reduced TRPV5 activity in human embryonic kidney cells transiently expressing the channel. Similarly, a strong inhibition of transepithelial Ca(2+) transport was observed after apical application of purified tTG to polarized rabbit CNT/CCD cells. Furthermore, tTG promoted the aggregation of the plasma membrane-associated fraction of TRPV5. Using patch clamp analysis, we observed a reduction in the pore diameter after tTG treatment, suggesting distinct structural changes in TRPV5 upon crosslinking by tTG. As N-linked glycosylation of TRPV5 is a key step in regulating channel function, we determined the effect of tTG in the N-glycosylation-deficient TRPV5 mutant. In the absence of N-linked glycosylation, TRPV5 was insensitive to tTG. Taken together, these observations imply that tTG is a novel extracellular enzyme inhibiting the activity of TRPV5. The inhibition of TRPV5 occurs in an N-glycosylation-dependent manner, signifying a common final pathway by which distinct extracellular factors regulate channel activity.01 maart 201