Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analyzing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAMM DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs

The ATP-Binding Cassette Proteins of the Deep-Branching Protozoan Parasite Trichomonas vaginalis

The ATP binding cassette (ABC) proteins are a family of membrane transporters and regulatory proteins responsible for diverse and critical cellular process in all organisms. To date, there has been no attempt to investigate this class of proteins in the infectious parasite Trichomonas vaginalis. We have utilized a combination of bioinformatics, gene sequence analysis, gene expression and confocal microscopy to investigate the ABC proteins of T. vaginalis. We demonstrate that, uniquely among eukaryotes, T. vaginalis possesses no intact full-length ABC transporters and has undergone a dramatic expansion of some ABC protein sub-families. Furthermore, we provide preliminary evidence that T. vaginalis is able to read through in-frame stop codons to express ABC transporter components from gene pairs in a head-to-tail orientation. Finally, with confocal microscopy we demonstrate the expression and endoplasmic reticulum localization of a number of T. vaginalis ABC transporters

The link between rejection sensitivity and borderline personality disorder:A systematic review and meta-analysis

OBJECTIVE: People with Borderline Personality Disorder (BPD) may experience heightened rejection sensitivity (RS), a disposition developing from repeated childhood rejecting experiences. It is not known whether the full RS model accounts for the cognitive-affective experiences common in BPD. This systematic review extends upon previous reviews, firstly by assessing the link between childhood rejecting experiences and adult RS, and secondly by considering the link between BPD and RS in both non-clinical and clinical samples.METHOD: Two research questions were devised, and searches based on predetermined criteria were conducted using PsycNET, PubMed, SCOPUS, and Web of Science. Data were extracted by one researcher and 20% was inter-rated, with high levels of agreement. Forty-three papers were systematically reviewed, and 31 included in meta-analysis and meta-regression.RESULTS: Studies assessing the link between childhood rejection and RS are limited; however, emotional abuse and neglect appears linked with RS. Pooled effect sizes suggest RS is linked with BPD (r = .326), with strong effect sizes when comparing clinical and control samples (r = .655). Qualitative synthesis suggests this may be mediated by executive control, although further research is required. The small number of studies considering the full RS model with regard to BPD suggests the interaction between emotional abuse and neglect affects rejection sensitivity; however, outcomes are inconsistent.CONCLUSIONS: Childhood rejection, particularly emotional abuse and neglect, appears to be linked to rejection sensitivity, and rejection sensitivity is linked to BPD. However, this may not be linear. Implications for clinical practice and research are discussed.PRACTITIONER POINTS: Rejection sensitivity is consistently linked with BPD, in clinical and non-clinical samples. Supporting mentalization or improved theory of mind may offer a therapeutic target for this disposition. Considering the causes and effects of rejection sensitivity may offer a non-blaming explanation of interpersonal difficulties in BPD and could be utilized as part of formulation and the therapeutic relationship. However, the possible interaction between emotional abuse and neglect and rejection sensitivity suggests rejection sensitivity is not always apparent for people with BPD. Idiosyncratic formulation should consider this. The literature included in the review is limited to Western populations with a high proportion of females, which may limit generalizability. Measures of rejection sensitivity included in the review were restricted to self-report, which may be subject to bias. Furthermore, measures of childhood rejection were retrospective in nature due to the exclusion of child samples. Further research should consider longitudinal and observational study designs.</p

Fine Tuning of Globin Gene Expression by DNA Methylation

Expression patterns in the globin gene cluster are subject to developmental regulation in vivo. While the γ(A) and γ(G) genes are expressed in fetal liver, both are silenced in adult erythrocytes. In order to decipher the role of DNA methylation in this process, we generated a YAC transgenic mouse system that allowed us to control γ(A) methylation during development. DNA methylation causes a 20-fold repression of γ(A) both in non-erythroid and adult erythroid cells. In erythroid cells this modification works as a dominant mechanism to repress γ gene expression, probably through changes in histone acetylation that prevent the binding of erythroid transcription factors to the promoter. These studies demonstrate that DNA methylation serves as an elegant in vivo fine-tuning device for selecting appropriate genes in the globin locus. In addition, our findings provide a mechanism for understanding the high levels of γ-globin transcription seen in patients with Hereditary Persistence of Fetal Hemoglobin, and help explain why 5azaC and butyrate compounds stimulate γ-globin expression in patients with β-hemoglobinopathies

Zentralnervöse Verarbeitung akustischer Informationen: Signaldetektion, Signallateralisation und zeitgebundene Informationsverarbeitung bei Patienten mit erworbenen Hirnschädigungen.

Die vorliegende Arbeit beschäftigt sich mit der Untersuchung der Funktionalität des zentral auditorischen Systems in Abhängigkeit von der Integrität einzelner hirnorganischer Strukturen, speziell bei Patienten mit erworbenen Hirnschädigungen. Es wird genauer auf die Verarbeitungsmechanismen, die für die Erkennung sprachlicher und nicht-sprachlicher Signale vonnöten sind eingegangen

The perception of prephonematic acoustic signals alters with age

Presently we are establishing two sets of psychoacoustic tests (basic tests and integrational tests) which should allow the differentiation of peripheral and central auditory processing in normal-hearing and hearing-impaired subjects. In a baseline study performed on one hundred, 20-70 years old normal-hearing subjects we used these tests to evaluate age-dependent alterations in auditory processing. The basic tests measured (1) the monaural audiogram, (2) the frequnecy resolution and the limits of simultaneous (3) frequency and (4) intensity discrimination. The integrational tests reported here evaluate the limits of binaural (5) frequency, (6) intensity and (7) temporal discrimination. These measures, together with tests under successive stimulus conditions, show that temporal processing and processing of stimulus frequency worsens with increasing age, while the processing of stimulus intensity remains stable