Transfection of murine LMTK− cells with purified HLA class I genes

The individual contributions of the first two external domains of the HLA-B7 heavy chain to the expression of allele-specific (B7) and locus-specific (B and C) antigenic determinants were investigated using hybrid class I genes. Hybrid genes were constructed in vitro by exon shuffling between the parent genes HLA-B7, HLA-Cw3, HLA-A3, and H-2K ^d, and their expression was monitored following transfection into mouse L cells. The results show that most allele-specific antigenic determinants are associated with the first external domain of the 137 heavy chain, whereas all the locus-specific antigenic determinants tested map to the second external domain.Abbreviations used in this paper BSA bovine serum albumin - FCS fetal calf serum - mAb monoclonal antibody - PBL peripheral blood lymphocytes - PBS phosphate-buffered saline     

Large-scale growth of the Plasmodium falciparum malaria parasite in a wave bioreactor

We describe methods for the large-scale in vitro culturing of synchronous and asynchronous blood-stage Plasmodium falciparum parasites in sterile disposable plastic bioreactors controlled by wave-induced motion (wave bioreactor). These cultures perform better than static flask cultures in terms of preserving parasite cell cycle synchronicity and reducing the number of multiple-infected erythrocytes. The straight-forward methods described here will facilitate the large scale production of malaria parasites for antigen and organelle isolation and characterisation, for the high throughput screening of compound libraries with whole cells or extracts, and the development of live- or whole-cell malaria vaccines under good manufacturing practice compliant standards.     

Words and Melody Are Intertwined in Perception of Sung Words: EEG and Behavioral Evidence

Language and music, two of the most unique human cognitive abilities, are combined in song, rendering it an ecological model for comparing speech and music cognition. The present study was designed to determine whether words and melodies in song are processed interactively or independently, and to examine the influence of attention on the processing of words and melodies in song. Event-Related brain Potentials (ERPs) and behavioral data were recorded while non-musicians listened to pairs of sung words (prime and target) presented in four experimental conditions: same word, same melody; same word, different melody; different word, same melody; different word, different melody. Participants were asked to attend to either the words or the melody, and to perform a same/different task. In both attentional tasks, different word targets elicited an N400 component, as predicted based on previous results. Most interestingly, different melodies (sung with the same word) elicited an N400 component followed by a late positive component. Finally, ERP and behavioral data converged in showing interactions between the linguistic and melodic dimensions of sung words. The finding that the N400 effect, a well-established marker of semantic processing, was modulated by musical melody in song suggests that variations in musical features affect word processing in sung language. Implications of the interactions between words and melody are discussed in light of evidence for shared neural processing resources between the phonological/semantic aspects of language and the melodic/harmonic aspects of music.     

Caspase-activated PAK-2 is regulated by subcellular targeting and proteasomal degradation

p21-activated protein kinases (PAKs) are a family of serine/threonine protein kinases that are activated by binding of the p21 G proteins Cdc42 or Rac. The ubiquitous PAK-2 (gamma-PAK) is unique among the PAK isoforms because it is also activated through proteolytic cleavage by caspases or caspase-like proteases. In response to stress stimulants such as tumor necrosis factor alpha or growth factor withdrawal, PAK-2 is activated as a full-length enzyme and as a proteolytic PAK-2p34 fragment. Activation of full-length PAK-2 stimulates cell survival, whereas proteolytic activation of PAK-2p34 is involved in programmed cell death. Here we provide evidence that the proapoptotic effect of PAK-2p34 is regulated by subcellular targeting and degradation by the proteasome. Full-length PAK-2 is localized in the cytoplasm, whereas the proteolytic PAK-2p34 fragment translocates to the nucleus. Subcellular localization of PAK-2 is regulated by nuclear localization and nuclear export signal motifs. A nuclear export signal motif within the regulatory domain prevents nuclear localization of full-length PAK-2. Proteolytic activation removes most of the regulatory domain and disrupts the nuclear export signal. The activated PAK-2p34 fragment contains a nuclear localization signal and translocates to the nucleus. However, levels of activated PAK-2p34 are tightly regulated through ubiquitination and degradation by the proteasome. Inhibition of degradation by blocking polyubiquitination results in significantly increased levels of PAK-2p34 and as a consequence, in stimulation of programmed cell death. Therefore, nuclear targeting and inhibition of degradation appear to be critical for stimulation of the cell death response by PAK-2p34.     

A new algorithm for computational image analysis of deformable motion at high spatial and temporal resolution applied to root growth. Roughly uniform elongation in the meristem and also,after an abrupt acceleration,in the elongation zone

A requirement for understanding morphogenesis is being able to quantify expansion at the cellular scale. Here, we present new software (RootflowRT) for measuring the expansion profile of a growing root at high spatial and temporal resolution. The software implements an image processing algorithm using a novel combination of optical flow methods for deformable motion. The algorithm operates on a stack of nine images with a given time interval between each (usually 10 s) and quantifies velocity confidently at most pixels of the image. The root does not need to be marked. The software calculates components of motion parallel and perpendicular to the local tangent of the root's midline. A variation of the software has been developed that reports the overall root growth rate versus time. Using this software, we find that the growth zone of the root can be divided into two distinct regions, an apical region where the rate of motion, i.e. velocity, rises gradually with position and a subapical region where velocity rises steeply with position. In both zones, velocity increases almost linearly with position, and the transition between zones is abrupt. We observed this pattern for roots of Arabidopsis, tomato (Lycopersicon lycopersicum), lettuce (Lactuca sativa), alyssum (Aurinia saxatilis), and timothy (Phleum pratense). These velocity profiles imply that relative elongation rate is regulated in a step-wise fashion, being low but roughly uniform within the meristem and then becoming high, but again roughly uniform, within the zone of elongation. The executable code for RootflowRT is available from the corresponding author on request.     

Sex differences in yolk hormones depend on maternal social status in Leghorn chickens (Gallus gallus domesticus)

Maternal hormones are known to be present in avian eggs and can have beneficial effects on chick development. Recently, differences in avian yolk steroid concentrations between the sexes have been demonstrated, and in this context steroids have been proposed to be part of the avian sex-determining mechanism. In our study, we show that it is very unlikely that androgen concentrations alone are the decisive part of the sex-determining mechanism. We found that sex-specific differences in the yolk hormones strongly depend on the social rank of the mother. First, dominant females, but not subdominant females, allocated significantly more testosterone to male eggs than to female eggs. Second, subordinate females increased the testosterone concentrations of female eggs. This pattern of yolk hormone deposition can be functionally explained. In polygynous species such as the chicken, reproductive success is more variable in males than in females. Parental investment in sons or daughters is therefore expected to occur in direct relation to parental rearing capacities. We found that the social status of a hen was indeed negatively correlated with her maternal capacities (for example, body mass, egg mass). Differential androgen deposition might thus provide a mechanism for adaptive maternal investment depending on both the sex of the egg and the social status of the mother.     

A novel functional class I lineage in zebrafish (Danio rerio), carp (Cyprinus carpio), and large barbus (Barbus intermedius) showing an unusual conservation of the peptide binding domains

Species from all major jawed vertebrate taxa possess linked polymorphic class I and II genes located in an MHC. The bony fish are exceptional with class I and II genes located on different linkage groups. Zebrafish (Danio rerio), common carp (Cyprinus carpio), and barbus (Barbus intermedius) represent highly divergent cyprinid genera. The genera Danio and Cyprinus diverged 50 million years ago, while Cyprinus and Barbus separated 30 million years ago. In this study, we report the first complete protein-coding class I ZE lineage cDNA sequences with high similarity between the three cyprinid species. Two unique complete protein-coding cDNA sequences were isolated in zebrafish, Dare-ZE*0101 and Dare-ZE*0102, one in common carp, Cyca-ZE*0101, and six in barbus, Bain-ZE*0101, Bain-ZE*0102, Bain-ZE*0201, Bain-ZE*0301, Bain-ZE*0401, and Bain-ZE*0402. Deduced amino acid sequences indicate that these sequences encode bonafide class I proteins. In addition, the presence of conserved potential peptide anchoring residues, exon-intron organization, ubiquitous expression, and polymorphism generated by positive selection on putative peptide binding residues support a classical nature of class I ZE lineage genes. Phylogenetic analyses revealed clustering of the ZE lineage clade with nonclassical cyprinid class I Z lineage clade away from classical cyprinid class I genes, suggesting a common ancestor of these nonclassical genes as observed for mammalian class I genes. Data strongly support the classical nature of these ZE lineage genes that evolved in a trans-species fashion with lineages being maintained for up to 100 million years as estimated by divergence time calculations.     

Testosterone, cuckoldry risk and extra-pair opportunities in the Seychelles warbler

In male birds, testosterone (T) plays an important role in aggressive and mate-attraction behaviour. In the cooperatively breeding Seychelles warbler, Acrocephalus sechellensis, extra-group copulations (EGCs) occur frequently, but are not accompanied by sexual courtship displays as in within-pair copulations. Paternity is nearly always gained by primary males. We investigated whether T levels and sperm storage capability (cloacal protuberance (CP)) in adult primary and subordinate males were related to timing of egg laying, levels of cuckoldry and extra-group paternity (EGP) opportunities. During the sexually active period before egg laying, T levels and CP were only elevated or enlarged (respectively) in primary males, and some suggestion was found that subordinate males do not invest in elevated T levels. The peak in T occurred during the fertile period of the female partner and corresponded to the peak period of male sexual displays and mate guarding, but was independent of cuckoldry risk (density of neighbouring primary males). CP was also enhanced during this period; however, CP but not T remained elevated after egg laying by their mates, and CP but not T was positively related to EGP opportunities (density of neighbouring fertile females). We conclude that T is involved in sexual courtship displays and mate guarding, but not in gaining EGCs. These findings contrast with those in other species where EGP involves elaborate sexual displays.