Monitoring lysosomal fusion in electrofused hybridoma cells

Dendritic and tumor cells are fused to produce hybridoma cells, which are considered to be used as cellular vaccines to treat cancer. Previous strategies for hybridoma cell production were based on the quantification of the electrofusion yield by labeling the cytoplasm of both parental cell types. However, a better physiological strategy would be to label subcellular structures related directly to the antigen presentation process. Therefore, we here electrofused the same amount of CHO cells stained with red and green fluorescent dextrans and have monitored the yield of hybridoma cell formation by measuring the fusion of red and green late endocytic organelles that are involved in antigen presentation. By using confocal microscopy, the level of fused, fluorescently labelled late endocytic compartments in a single hybridoma cell was determined. The results demonstrate that organellar fusion occurs in hybridomas, which is time- and temperature-dependent. This approach therefore provides a new method for the hybridoma cell vaccine evaluation, which is based on the intracellular physiological mechanism of antigen presentation.     

Acquired obesity is associated with changes in the serum lipidomic profile independent of genetic effects--a monozygotic twin study

Both genetic and environmental factors are involved in the etiology of obesity and the associated lipid disturbances. We determined whether acquired obesity is associated with changes in global serum lipid profiles independent of genetic factors in young adult monozygotic (MZ) twins. 14 healthy MZ pairs discordant for obesity (10 to 25 kg weight difference) and ten weight concordant control pairs aged 24-27 years were identified from a large population-based study. Insulin sensitivity was assessed by the euglycemic clamp technique, and body composition by DEXA (% body fat) and by MRI (subcutaneous and intra-abdominal fat). Global characterization of lipid molecular species in serum was performed by a lipidomics strategy using liquid chromatography coupled to mass spectrometry. Obesity, independent of genetic influences, was primarily related to increases in lysophosphatidylcholines, lipids found in proinflammatory and proatherogenic conditions and to decreases in ether phospholipids, which are known to have antioxidant properties. These lipid changes were associated with insulin resistance, a pathogonomic characteristic of acquired obesity in these young adult twins. Our results show that obesity, already in its early stages and independent of genetic influences, is associated with deleterious alterations in the lipid metabolism known to facilitate atherogenesis, inflammation and insulin resistance.     

Transition dynamics and selection of the distinct S-DNA and strand unpeeling modes of double helix overstretching

Recent studies have revealed two distinct pathways for the DNA overstretching transition near 65 pN: 'unpeeling' of one strand from the other, and a transition from B-DNA to an elongated double-stranded 'S-DNA' form. However, basic questions concerning the dynamics of these transitions, relative stability of the two competing overstretched states, and effects of nicks and free DNA ends on overstretching, remain open. In this study we report that: (i) stepwise extension changes caused by sequence-defined barriers occur during the strand-unpeeling transition, whereas rapid, sequence-independent extension fluctuations occur during the B to S transition; (ii) the secondary transition that often occurs following the overstretching transition is strand-unpeeling, during which the extension increases by 0.01-0.02 nm per base pair of S-DNA converted to single-stranded DNA at forces between 75 and 110 pN; (iii) even in the presence of nicks or free ends, S-DNA can be stable under physiological solution conditions; (iv) distribution of small GC-rich islands in a large DNA plays a key role in determining the transition pathways; and (v) in the absence of nicks or free ends, torsion-unconstrained DNA undergoes the overstretching transition via creation of S-DNA. Our study provides a new, high-resolution understanding of the competition between unpeeling and formation of S-DNA.     

In vitro tests within the REACH information strategies

Tonnage-based information requirements are specified in the proposal on the regulation on the Registration, Evaluation and Authorisation of Chemicals (REACH) in the European Union. The hazard assessment for toxic endpoints should be performed by using a tiered approach, i.e. as an information strategy (IS), starting with an evaluation of all of the data already available, including animal in vivo and in vitro data, and human evidence and case reports, as well as data from (Quantitative)-Structure Activity Relationships ([Q]SARs) or read-across, before any further testing is suggested. To contribute to the implementation of the REACH system, the Nordic countries launched two projects: 1) a review of currently used testing strategies, including a comparison with the REACH requirements; and 2) the development of detailed ISs for skin and eye irritation/corrosion. The review showed that the ISs and classification criteria for the selected endpoints are inconsistent in many cases. In the classification criteria, human data and in vivo test results are usually the prerequisites. Other types of information, such as data from in vitro studies, can sometimes be used, but usually as supportive evidence only. This differs from the REACH ISs, where QSARs, read-across and in vitro testing are important elements. In the other part of the project, an IS for skin and eye irritation/corrosion was proposed. The strategy was "tested" by using four high production volume (HPV) chemicals: hydrogen peroxide, methyl tertiary-butyl ether (MTBE), trivalent chromium, and diantimony trioxide, but only MTBE and trivalent chromium are dealt with in this paper. The "test" revealed that in vivo data, human case reports and physical-chemical data were available and could be used in the evaluation. Classification could be based on the proposed IS and the existing data in all cases, except for the eye irritation/corrosion of trivalent chromium. Weight-of-evidence analysis appeared to be a useful step in the ISs proposed, and including it in the REACH strategies should be considered. For these chemicals, few in vitro and (Q)SAR data were available--more of these data would be generated, if the relevant guidance and legislation on classification were updated.     

A multi-criteria decision-making model for classifying wood products with respect to their impact on environment

Background, aim and scope  

Although life cycle assessment is frequently used in scientific studies of product comparison, many practitioners are looking for improvements in the normalisation, grouping and weighting of life cycle inventory results. Local conditions, which are well known to local experts, are very important to these steps. The goal of this work was to develop a computer-based decision support system for classifying wood products according to their influence on the environment in their whole life cycle. The model specifically addresses local conditions in the Republic of Slovenia and was developed by Slovenian experts.     

Quantification of lysophosphatidic acids in rat brain tissue by liquid chromatography–electrospray tandem mass spectrometry

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological functions. A highly selective and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for the determination of LPAs (16:0 LPA, 18:0 LPA, 18:1 LPA, 20:4 LPA) in rat brain cryosections. After partitioning the LPAs from other lipophilic material present in the tissue with a liquid–liquid extraction, a reversed-phase column and ion pair technique was used for separating analytes with a gradient elution. An internal standard (17:0 LPA) was included in the analysis. Detection and quantification of the LPAs were carried out with a triple quadrupole mass spectrometer using negative electrospray ionization (ESI) and multiple reaction monitoring (MRM). The artificial formation of LPAs from lysophosphatidylcholines during the sample preparation procedure and instrumentation was carefully studied during the method development. The method was validated; acceptable selectivity, accuracy, precision, recovery, and stability were obtained for concentrations within the calibration curve range of 0.02–1.0 μM for LPAs. The quantification limit of the assay was 54 fmol injected into column for each LPAs. The method was applied to comparative studies of LPA levels in rat brain cryosections after the various chemical pre-treatments of the sections.     

Genetic Variation and the Role of Insect Life History Traits in the Ability of Drosophila Larvae to Develop in the Presence of a Competing Filamentous Fungus

Competitive interactions between organisms from distantly related phylogenetical branches have been suggested as being one of the most pervasive forms of interspecific competition. However, so-called inter-kingdom competition has rarely been the focus of ecological and evolutionary studies. Thus, a relatively novel hypothesis has been proposed on the basis that saprophagous insects might intensively compete with filamentous fungi for ephemeral resources (e.g. decaying plant tissue). Consideration that life history traits (e.g. developmental time) are adaptive in determining developmental success in the presence of con- or hetero-specifics competitors implies that these traits have been progressively established by natural selection. Because a similar scenario may apply to antagonistic interactions between saprophagous insects and filamentous fungi, one can expect the existence of heritable variation in developmental success when insect larvae are forced to grow in the presence of noxious mould. Therefore, this study aimed at discovering whether a local population of Drosophila melanogaster indeed harbours genetic variation in developmental success in the presence of the mould Aspergillus niger. By using the isofemale line technique, single larvae forced to feed on fungal infected or uninfected substrate were analysed for variation in survival probability to the adult stage, developmental time and body size of emerged adults. I found genetic variation in survival probability in fungal infected substrates but not in uninfected larval food sources. Mean developmental time and body size varied significantly among isofemale lines in both types of larval environment. Survival was negatively correlated with developmental time on fungal infected substrate, but variation in developmental time on fungal-free substrates was not correlated with survival on fungal infected food patches. Within-trait correlation between fungal infected and uninfected substrates was surprisingly weak, and developmental time was not correlated with body size. The results of this study demonstrate (a) the existence of genetic variation for larval developmental success in the presence of A. niger in a Drosophila population, and (b) heritability of important insect life history traits differed as a function of the larval environment (fungal infected or uninfected feeding substrate). I discuss models that might explain heritability differences and the evolutionary consequences of these results.     

Integrin trafficking regulated by Rab21 is necessary for cytokinesis

Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer.