Molecular evolution of the MAGUK family in metazoan genomes

Background  

Development, differentiation and physiology of metazoans all depend on cell to cell communication and subsequent intracellular signal transduction. Often, these processes are orchestrated via sites of specialized cell-cell contact and involve receptors, adhesion molecules and scaffolding proteins. Several of these scaffolding proteins important for synaptic and cellular junctions belong to the large family of membrane-associated guanylate kinases (MAGUK). In order to elucidate the origin and the evolutionary history of the MAGUKs we investigated full-length cDNA, EST and genomic sequences of species in major phyla.     

Darwin and His Pigeons. The Analogy Between Artificial and Natural Selection Revisited

The analogy between artificial selection of domestic varieties and natural selection in nature was a vital element of Darwin’s argument in his Origin of Species. Ever since, the image of breeders creating new varieties by artificial selection has served as a convincing illustration of how the theory works. In this paper I argue that we need to reconsider our understanding of Darwin’s analogy. Contrary to what is often assumed, nineteenth-century animal breeding practices constituted a highly controversial field that was fraught with difficulties. It was only with considerable effort that Darwin forged his analogy, and he only succeeded by downplaying the importance of two other breeding techniques – crossing of varieties and inbreeding – that many breeders deemed essential to obtain new varieties. Part of the explanation for Darwin’s gloss on breeding practices, I shall argue, was that the methods of his main informants, the breeders of fancy pigeons, were not representative of what went on in the breeding world at large. Darwin seems to have been eager to take the pigeon fanciers at their word, however, as it was only their methods that provided him with the perfect analogy with natural selection. Thus while his studies of domestic varieties were important for the development of the concept of natural selection, the reverse was also true: Darwin’s comprehension of breeding practices was moulded by his understanding of the working of natural selection in nature. Historical studies of domestic breeding practices in the eighteenth and nineteenth century confirm that, besides selection, the techniques of inbreeding and crossing were much more important than Darwin’s interpretation allowed for. And they still are today. This calls for a reconsideration of the pedagogic use of Darwin’s analogy too.     

Developmental experience alters information coding in auditory midbrain and forebrain neurons

In songbirds, species identity and developmental experience shape vocal behavior and behavioral responses to vocalizations. The interaction of species identity and developmental experience may also shape the coding properties of sensory neurons. We tested whether responses of auditory midbrain and forebrain neurons to songs differed between species and between groups of conspecific birds with different developmental exposure to song. We also compared responses of individual neurons to conspecific and heterospecific songs. Zebra and Bengalese finches that were raised and tutored by conspecific birds, and zebra finches that were cross‐tutored by Bengalese finches were studied. Single‐unit responses to zebra and Bengalese finch songs were recorded and analyzed by calculating mutual information (MI), response reliability, mean spike rate, fluctuations in time‐varying spike rate, distributions of time‐varying spike rates, and neural discrimination of individual songs. MI quantifies a response's capacity to encode information about a stimulus. In midbrain and forebrain neurons, MI was significantly higher in normal zebra finch neurons than in Bengalese finch and cross‐tutored zebra finch neurons, but not between Bengalese finch and cross‐tutored zebra finch neurons. Information rate differences were largely due to spike rate differences. MI did not differ between responses to conspecific and heterospecific songs. Therefore, neurons from normal zebra finches encoded more information about songs than did neurons from other birds, but conspecific and heterospecific songs were encoded equally. Neural discrimination of songs and MI were highly correlated. Results demonstrate that developmental exposure to vocalizations shapes the information coding properties of songbird auditory neurons. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 70: 235–252, 2010.     

Heat pretreatment increases resolution in DNA flow cytometry of paraffin-embedded tumor tissue

BACKGROUND: Flow cytometry of single-cell suspensions prepared by enzymatic digestion from formalin-fixed, paraffin-embedded tissue suffers from several major drawbacks. The most important factors that influence the results are the high and unpredictable coefficients of variation (CVs) of the G0/G1 peak in the DNA histogram and reduction of propidium iodide (PI) intercalation with DNA, resulting from protein cross-linking by formalin. METHODS: In this study we introduce a heating step (2 h incubation in citrate solution at 80 degrees C) prior to a brief pepsin digestion of tissue sections in the protocol for DNA content analysis of formalin-fixed and paraffin-embedded tissue. This new method is compared with established methods for the preparation of cell suspensions from frozen and paraffin-embedded tissues with respect to cell yield, DNA histogram resolution, DNA dye saturation kinetics, cell cycle parameters, and antigen retrieval in various epithelial and nonepithelial tissues. RESULTS: The recovery of single cells from the paraffin sections was doubled by the heat treatment step, while the limited time of proteolysis resulted in decreased cell debris. Furthermore, an increased fraction of cells became cytokeratin-positive, while these immunocytochemically stained cells also exhibited a higher mean fluorescence intensity. The DNA histograms prepared from cell suspensions obtained according to this new protocol showed a significantly improved resolution, leading to a better identification of peridiploid cell populations. Heat pretreatment of paraffin-embedded archival tissue sections showed PI saturation kinetics similar to, or even better than, those of fresh unfixed tissues, independent of duration of fixation. CONCLUSIONS: This new method, making use of routinely available antigen retrieval principles, thus allows high-resolution DNA analysis of routinely fixed and paraffin-embedded tissue samples. Using external reference cells, inter- and intralaboratory standardization of DNA histograms can be achieved.     

Mitochondrial growth and division during the cell cycle in HeLa cells

The growth and division of mitochondria during the cell cycle was investigated by a morphometric analysis of electron micrographs of synchronized HeLa cells. The ratio of total outer membrane contour length to cytoplasmic area did not vary significantly during the cell cycle, implying a continuous growth of the mitochondrial outer membrane. The mean fraction of cytoplasmic area occupied by mitochondrial profiles was likewise found to remain constant, indicating that the increase in total mitochondrial volume per cell occurs continuously during interphase, in such a way that the mitochondrial complement occupies a constant fraction( approximately 10-11(percent)) of the volume of the cytoplasm. The mean area, outer membrane contour length, and axis ratio of the mitochondrial profiles also did not vary appreciably during the cell cycle; furthermore, the close similarity of the frequency distributions of these parameters for the six experimental time-points suggested a stable mitochondrial shape distribution. The constancy of both the mean mitochondrial profile area and the number of mitochondrial profiles per unit of cytoplasmic area was interpreted to indicate the continuous division of mitochondria at the level of the cell population. Furthermore, no evidence was found for the occurrence of synchronous mitochondrial growth and division within individual cells. Thus, it appears that, in HeLa cells, there is no fixed temporal relationship between the growth and division of mitochondria and the events of the cell cycle. A number of statistical methods were developed for the purpose of making numerical estimates of certain three-dimensional cellular and mitochondrial parameters. Mean cellular and cytoplasmic volumes were calculated for the six time-points; both exhibited a nonlinear, approx. twofold increase. A comparison of the axis ratio distributions of the mitochondrial profiles with theoretical distributions expected from random sectioning of bodies of various three-dimensional shapes allowed the derivation of an "average" mitochondrial shape. This, in turn, permitted calculations to be made which expressed the two-dimensional results in three-dimensional terms. Thus, the estimated values for the number of mitochondria per unit of cytoplasmic volume and for the mean mitochondrial volume were found to remain constant during the cell cycle, while the estimated number of mitochondria per cell increase approx. twofold in an essentially continuous manner.     

Global expression profiling of theophylline response genes in macrophages: evidence of airway anti-inflammatory regulation

Background

Theophylline has been used widely as a bronchodilator for the treatment of bronchial asthma and has been suggested to modulate immune response. While the importance of macrophages in asthma has been reappraised and emphasized, their significance has not been well investigated. We conducted a genome-wide profiling of the gene expressions of macrophages in response to theophylline.

Methods

Microarray technology was used to profile the gene expression patterns of macrophages modulated by theophylline. Northern blot and real-time quantitative RT-PCR were also used to validate the microarray data, while Western blot and ELISA were used to measure the levels of IL-13 and LTC4.

Results

We identified dozens of genes in macrophages that were dose-dependently down- or up-regulated by theophylline. These included genes related to inflammation, cytokines, signaling transduction, cell adhesion and motility, cell cycle regulators, and metabolism. We observed that IL-13, a central mediator of airway inflammation, was dramatically suppressed by theophylline. Real-time quantitative RT-PCR and ELISA analyses also confirmed these results, without respect to PMA-treated THP-1 cells or isolated human alveolar macrophages. Theophylline, rolipram, etazolate, db-cAMP and forskolin suppressed both IL-13 mRNA expression (~25%, 2.73%, 8.12%, 5.28%, and 18.41%, respectively) and protein secretion (<10% production) in macrophages. These agents also effectively suppressed LTC4 expression.

Conclusion

Our results suggest that the suppression of IL-13 by theophylline may be through cAMP mediation and may decrease LTC4 production. This study supports the role of theophylline as a signal regulator of inflammation, and that down regulation of IL-13 by theophylline may have beneficial effects in inflammatory airway diseases.     

Breeding Without Mendelism: Theory and Practice of Dairy Cattle Breeding in the Netherlands 1900–1950

In the 1940s and 1950s, Dutch scientists became increasingly critical of the practices of commercial dairy cattle breeders. Milk yields had hardly increased for decades, and the scientists believed this to be due to the fact that breeders still judged the hereditary potential of their animals on the basis of outward characteristics. An objective verdict on the qualities of breeding stock could only be obtained by progeny testing, the scientists contended: the best animals were those that produced the most productive offspring. Some scientists had been making this claim since the beginning of the twentieth century. Why was it that their advice was apparently not heeded by breeders for so long? And what were the methods and beliefs that guided their practices? In this paper I intend to answer these questions by analysing the practical realities of dairy farming and stock breeding in The Netherlands between 1900 and 1950. Breeders continued to employ traditional breeding methods that had proven their effectiveness since the late eighteenth century. Their methods consisted in inbreeding – breeding in ?bloodlines,’ as they called it – and selection on the basis of pedigree, conformation and milk recording data. Their aims were ?purity’ and ?uniformity’ of type. Progeny testing was not practiced due to practical difficulties. Before World War II, scientists acknowledged that genetic theory was of little practical use to breeders of livestock. Still, hereditary theory was considered to be helpful to assess the value of the breeders’ methods. For instance, striving for purity was deemed to be consistent with Mendelian theory. Yet the term purity had different connotations for scientists and practical workers. For the former, it referred to homozygosity; for the latter, it rather buttressed the constancy of a distinct commercial ?brand.’ Until the 1940s, practical breeders and most scientists were agreed that selecting animals purely for production was ill-advised. Cows of the extreme dairy type were believed to be prone to bovine tuberculosis. This conviction was at the basis of the development of ?the modern Friesian,’ a rather robust type of dairy cow that was also valued for its aesthetically pleasing conformation and that became a commercial success. Contrary to the scientists’ claims, it was not only for commercial reasons that breeders were reluctant to give up their modern Friesians after World War II, when the introduction of artificial insemination opened up the possibility of breeding more productive types by means of progeny testing. The political economy of breeding did indeed require breeders to protect their breed as a recognisable brand. Yet the moral economy of breeding must also be taken into account: the modern Friesian was also a product of widely shared normative standards of good and responsible farming.     

Why Wait? Early Determinants of School Dropout in Preventive Pediatric Primary Care

Background

To answer the question of what bio-psychosocial determinants in infancy, early and middle childhood, and adolescence predict school drop-out in young adulthood, we approached the complex process towards school dropout as a multidimensional, life-course phenomenon. The aim is to find signs of heightened risks of school dropout as early as possible which will eventually help public health workers in reducing these risks.

Methods

In a case-control design, we used data from both the Preventive Pediatric Primary Care (PPPC) files (that contain information from birth onwards) and additional questionnaires filled out by 529 youngsters, aged 18–23 years, and living in the South-east of the Netherlands. We first conducted univariate logistic regression analyses with school-dropout as the dependent variable. Backward and forward stepwise analyses with the significant variables were done with variables pertaining to the 0 to 4 year period. Remaining significant variables were forced into the next model and subsequently variables pertaining to respectively the 4 to 8, 8 to 12 and 12 to 16 year period were introduced in a stepwise analysis. All analyses were cross-validated in an exploratory and confirmatory random half of the sample.

Results

One parent families and families with a non-Western background less often attended the health examinations of the PPPC and such less attendance was related to school dropout. The birth of a sibling (OR 0.63, 95% CI 0.43–0.93) in infancy and self-efficacy (OR 0.53, 95% CI 0.38–0.74) in adolescence decreased the odds of school dropout; externalizing behavior (OR 2.81, 95% CI 1.53–5.14) in middle childhood and (sickness) absence (OR 5.62, 95% CI 2.18–14.52) in adolescence increased the risks.

Conclusion

To prevent school dropout, PPPC professionals should not wait until imminent dropout, but should identify and tackle risk factors as early as possible and actively approach youngsters who withdraw from public health care.