Hyperosmosis enhances radiation and hydroxyurea resistance of Schizosaccharomyces pombe checkpoint mutants through the spindle checkpoint and delayed cytokinesis

The DNA damage and stress response pathways interact to regulate cellular responses to genotoxins and environmental stresses. How these pathways interact in Schizosaccharomyces pombe is not well understood. We demonstrate that osmotic stress suppresses the DNA damage sensitivity of checkpoint mutants, and that this occurs through three distinct cell cycle delays. A delay in G2/M is dependent on Srk1. Progression through mitosis is halted by the Mad2‐dependent spindle checkpoint. Finally, cytokinesis is impaired by modulating Cdc25 expression. These three delays, imposed by osmotic stress, together compensate for the loss of checkpoint signalling.     

Chiral symmetry breaking: Experimental results and computer analysis of a liquid-phase autoxidation

The autoxidation of tetralin is treated as a model reaction system to define the applicability of stereospecific autocatalysis. This concept, predicting a spontaneous amplification of enantiomeric excess generated by an autocatalytic chemical reaction, is used in several theoretical models as an explanation for the origin of natural optical activity. The reaction system investigated obeys the basic criteria of these models: a chiral intermediate (tetralin hydroperoxide) is produced from an achiral substrate (tetralin) via an autocatalytic pathway where the feedback mechanism is expected to generate a state of broken chiral symmetry. In order to test the amplification capacity of this reaction a computer analysis of the kinetic scheme is performed. This simulation is derived from the known kinetic scheme of autoxidation and is validated by fitting the experimentally observed data of hydroperoxide evolution. Calculations show that this model allows powerful amplification of enantiomeric excess and a transient amplification of the optical rotation. It is also demonstrated that the model system exhibits pronounced sensitivity toward any loss of absolute configuration of the involved chiral species. Since an amplification effect results exclusively at a high degree of stereoselectivity, it is concluded that stereospecific autocatalysis is possible in systems which show template reactions, crystallization, or colloidal effects. © 1993 Wiley-Liss, Inc.     

New Approaches for Absolute Quantification of Stable‐Isotope‐Labeled Peptide Standards for Targeted Proteomics Based on a UV Active Tag

Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re‐quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.     

German Translation and Validation of the Cognitive Style Questionnaire Short Form (CSQ-SF-D)

Background

The Cognitive Style Questionnaire is a valuable tool for the assessment of hopeless cognitive styles in depression research, with predictive power in longitudinal studies. However, it is very burdensome to administer. Even the short form is still long, and neither this nor the original version exist in validated German translations.

Methods

The questionnaire was translated from English to German, back-translated and commented on by clinicians. The reliability, factor structure and external validity of an online form of the questionnaire were examined on 214 participants. External validity was measured on a subset of 90 subjects.

Results

The resulting CSQ-SF-D had good to excellent reliability, both across items and subscales, and similar external validity to the original English version. The internality subscale appeared less robust than other subscales. A detailed analysis of individual item performance suggests that stable results could be achieved with a very short form (CSQ-VSF-D) including only 27 of the 72 items.

Conclusions

The CSQ-SF-D is a validated and freely distributed translation of the CSQ-SF into German. This should make efficient assessment of cognitive style in German samples more accessible to researchers.     

Equatorial segment protein defines a discrete acrosomal subcompartment persisting throughout acrosomal biogenesis

The equatorial segment of the acrosome underlies the domain of the sperm that fuses with the egg membrane during fertilization. Equatorial segment protein (ESP), a novel 349-amino acid concanavalin-A-binding protein encoded by a two-exon gene (SP-ESP) located on chromosome 15 at q22, has been localized to the equatorial segment of ejaculated human sperm. Light microscopic immunofluorescent observations revealed that during acrosome biogenesis ESP first appears in the nascent acrosomal vesicle in early round spermatids and subsequently segregates to the periphery of the expanding acrosomal vesicle, thereby defining a peripheral equatorial segment compartment within flattened acrosomal vesicles and in the acrosomes of early and late cap phase, elongating, and mature spermatids. Electron microscopic examination revealed that ESP segregates to an electron-lucent subdomain of the condensing acrosomal matrix in Golgi phase round spermatids and persists in a similar electron-lucent subdomain within cap phase spermatids. Subsequently, ESP was localized to electron-dense regions of the equatorial segment and the expanded equatorial bulb in elongating spermatids and mature sperm. ESP is the earliest known protein to be recognized as a marker for the specification of the equatorial segment, and it allows this region to be traced through all phases of acrosomal biogenesis. Based on these observations, we propose a new model of acrosome biogenesis in which the equatorial segment is defined as a discrete domain within the acrosomal vesicle as early as the Golgi phase of acrosome biogenesis.     

Kinetic analysis of artificial peptide self-replication. Part I: the homochiral case

Computational kinetic analysis of a lately discovered homochiral peptide self-replicator is presented. A 6-step kinetic model was designed that addresses the main reactions and hydrophobic interactions involved in this template-directed, autocatalytic system and that gave rise to excellent fitting of 4 previously published independent experimental series. The model sheds light on the mechanistic principle of the reaction system and illustrates directly a number of dynamic properties such as the observed autocatalytic efficiency. It was found that the dynamics are basically governed by two reversible hydrophobic interactions: between the template and a peptide fragment and between two template species. The later association was determined to be considerably more favored, which leads to the predominant presence of the catalytically inactive template dimer in the reaction system. Our results show that the involvement of a template trimer is not necessary to obtain the observed fittings.     

Insulin-like growth factors and binding proteins in early milk from mothers of preterm and term infants

Breast-fed preterm infants often show a better outcome, partly ascribed to the benefit of insulin-like growth factors (IGFs) and their binding proteins (IGFBP). We compared IGF-I, IGF-II, IGFBP-2 and IGFBP-3 levels, measured by radioimmunoassays in milk samples from 30 mothers of preterm (<31 weeks) and from 19 mothers of term (>37 weeks) infants at days 7 and 21 postpartum. Proteolysis of IGFBP-2 within mother's milk and digestion of (125)I-IGF-II and (125)I-IGFBP-2 by gastric juice from neonates were assessed by electrophoretic techniques. Mean concentrations did not differ between preterm and term milk: IGF-I (2.8 +/- 0.2 vs. 2.3 +/- 0.1 ng/ml), IGF-II (12.0 +/- 0.4 vs. 12.2 +/- 0.5 ng/ml), IGFBP-3 (100.0 +/- 5.1 vs. 80.0 +/- 5.8 ng/ml), but did so for IGFBP-2 (3,144 +/- 172 vs. 2,428 +/- 188 ng/ml, p < 0.02). Immunoblots revealed 42% (p < 0.05) more IGFBP-2 fragments of 14 and 25 kDa in preterm milk. Incubation with gastric juice caused cleavage of (125)I-IGFBP-2 and partial cleavage of (125)I-IGF-II. Mutual complexation protected IGF-II and IGFBP-2 from cleavage, suggesting that both are likely to arrive in the bowel in an intact form to exert promotive effects. The results provide further evidence that IGFBP-2 and IGF-II in breast milk are relevant factors for the early development of preterm infants.