Ribosomal RNA genes in Scots pine (Pinus sylvestris L.): Chromosomal organization and structure

The nuclear 18S, 5.8S and 25S rRNA genes exist as thousands of rDNA repeats in the Scots pine genome. The number and location of rDNA loci (nucleolus organizers, NORs) were studied by cytological methods, and a restriction map from the coding region of the Scots pine rDNA repeat was constructed using digoxigenin-labeled flax rDNA as a probe. Based on the maximum number of nucleoli and chromosomal secondary constrictions, Scots pine has at least eight NORs in its haploid genome. The size of the Scots pine rDNA repeat unit is approximately 27 kb, two- or threefold larger than the typical angiosperm rDNA unit, but similar in size to other characterized conifer rDNA repeats. The intergenic spacer region (IGS) of the rDNA repeat unit in Scots pine is longer than 20 kb, and the transcribed spacer regions surrounding the 5.8S gene (ITS1 and ITS2) span a region of 2.9 kb. Restriction analysis revealed that although the coding regions of rDNA repeats are homogeneous, heterogeneity exists in the intergenic spacer region between individuals, as well as among the rDNA repeats within individuals.     

Studying genetics of adaptive variation in model organisms: flowering time variation in Arabidopsis lyrata

Arabidopsis thaliana has emerged as a model organism for plant developmental genetics, but it is also now being widely used for population genetic studies. Outcrossing relatives of A. thaliana are likely to provide suitable additional or alternative species for studies of evolutionary and population genetics. We have examined patterns of adaptive flowering time variation in the outcrossing, perennial A. lyrata. In addition, we examine the distribution of variation at marker genes in populations form North America and Europe. The probability of flowering in this species differs between southern and northern populations. Northern populations are much less likely to flower in short than in long days. A significant daylength by region interaction shows that the northern and southern populations respond differently to the daylength. The timing of flowering also differs between populations, and is made shorter by long days, and in some populations, by vernalization. North American and European populations show consistent genetic differentiation over microsatellite and isozyme loci and alcohol dehydrogenase sequences. Thus, the patterns of variation are quite different from those in A. thaliana, where flowering time differences show little relationship to latitude of origin and the genealogical trees of accessions vary depending on the genomic region studied. The genetic architecture of adaptation can be compared in these species with different life histories.     

Contractility and structure of adult rat seminiferous tubules in organ culture

Summary Isolated pieces of seminiferous tubules of adult rats were grown in organ culture for up to 8 weeks in Petri dishes on the surface of nutrient agar. The medium consisted of newborn calf serum, Eagle's minimum essential medium, glutamate and antibiotics. This method allowed observation of the contractions of the seminiferous tubules in the culture. Contractility, light and electron microscopic structure and histochemically demonstrable activities of alkaline phosphatase and ATPase of the tubule walls were studied at 1-week intervals. The contractility and alkaline phosphatase activity were maintained in the tubule wall for 3 weeks, and the activity of ATPase was maintained for 5 weeks. The thin filaments of the myoid cells, which are responsible for the contractility, were seen with the electron microscope in tubules cultured for 5 weeks. The organ culture method described in the present paper seems to be valuable for studies concerning the functioning of the myoid cells of the seminiferous tubules and the possibility that this is regulated by hormones.     

Complex Genetic Effects on Early Vegetative Development Shape Resource Allocation Differences Between Arabidopsis lyrata Populations

Costs of reproduction due to resource allocation trade-offs have long been recognized as key forces in life history evolution, but little is known about their functional or genetic basis. Arabidopsis lyrata, a perennial relative of the annual model plant A. thaliana with a wide climatic distribution, has populations that are strongly diverged in resource allocation. In this study, we evaluated the genetic and functional basis for variation in resource allocation in a reciprocal transplant experiment, using four A. lyrata populations and F₂ progeny from a cross between North Carolina (NC) and Norway parents, which had the most divergent resource allocation patterns. Local alleles at quantitative trait loci (QTL) at a North Carolina field site increased reproductive output while reducing vegetative growth. These QTL had little overlap with flowering date QTL. Structural equation models incorporating QTL genotypes and traits indicated that resource allocation differences result primarily from QTL effects on early vegetative growth patterns, with cascading effects on later vegetative and reproductive development. At a Norway field site, North Carolina alleles at some of the same QTL regions reduced survival and reproductive output components, but these effects were not associated with resource allocation trade-offs in the Norway environment. Our results indicate that resource allocation in perennial plants may involve important adaptive mechanisms largely independent of flowering time. Moreover, the contributions of resource allocation QTL to local adaptation appear to result from their effects on developmental timing and its interaction with environmental constraints, and not from simple models of reproductive costs.     

Modified Lipoprotein-Derived Lipid Particles Accumulate in Human Stenotic Aortic Valves

In aortic stenosis plasma lipoprotein-derived lipids accumulate in aortic valves. Here, we first compared the lipid compositions of stenotic aortic valves and atherosclerotic plaque cores. Both pathological tissues were found to be enriched in cholesteryl linoleate, a marker of extracellularly accumulated lipoproteins. In addition, a large proportion of the phospholipids were found to contain arachidonic acid, the common precursor of a number of proinflammatory lipid mediators. Next, we isolated and characterized extracellular lipid particles from human stenotic and non-stenotic control valves, and compared them to plasma lipoproteins from the same subjects. The extracellular valvular lipid particles were isolated from 15 stenotic and 14 non-stenotic aortic valves. Significantly more apoB-100-containing lipid particles were found in the stenotic than in the non-stenotic valves. The majority of the lipid particles isolated from the non-stenotic valves had sizes (23±6.2 nm in diameter) similar to those of plasma low density lipoprotein (LDL) (22±1.5 nm), while the lipid particles from stenotic valves were not of uniform size, their sizes ranging from 18 to more than 500 nm. The lipid particles showed signs of oxidative modifications, and when compared to isolated plasma LDL particles, the lipid particles isolated from the stenotic valves had a higher sphingomyelin/phosphatidylcholine –ratio, and also higher contents of lysophosphatidylcholine and unesterified cholesterol. The findings of the present study reveal, for the first time, that in stenotic human aortic valves, infiltrated plasma lipoproteins have undergone oxidative and lipolytic modifications, and become fused and aggregated. The generated large lipid particles may contribute to the pathogenesis of human aortic stenosis.     

Revealing the True Incidence of Pandemic A(H1N1)pdm09 Influenza in Finland during the First Two Seasons — An Analysis Based on a Dynamic Transmission Model

The threat of the new pandemic influenza A(H1N1)pdm09 imposed a heavy burden on the public health system in Finland in 2009-2010. An extensive vaccination campaign was set up in the middle of the first pandemic season. However, the true number of infected individuals remains uncertain as the surveillance missed a large portion of mild infections. We constructed a transmission model to simulate the spread of influenza in the Finnish population. We used the model to analyse the two first years (2009-2011) of A(H1N1)pdm09 in Finland. Using data from the national surveillance of influenza and data on close person-to-person (social) contacts in the population, we estimated that 6% (90% credible interval 5.1 – 6.7%) of the population was infected with A(H1N1)pdm09 in the first pandemic season (2009/2010) and an additional 3% (2.5 – 3.5%) in the second season (2010/2011). Vaccination had a substantial impact in mitigating the second season. The dynamic approach allowed us to discover how the proportion of detected cases changed over the course of the epidemic. The role of time-varying reproduction number, capturing the effects of weather and changes in behaviour, was important in shaping the epidemic.     

Demographic history has influenced nucleotide diversity in European Pinus sylvestris populations

To infer the role of natural selection in shaping standing genetic diversity, it is necessary to assess the genomewide impact of demographic history on nucleotide diversity. In this study we analyzed sequence diversity of 16 nuclear loci in eight Pinus sylvestris populations. Populations were divided into four geographical groups on the basis of their current location and the geographical history of the region: northern Europe, central Europe, Spain, and Turkey. There were no among-group differences in the level of silent nucleotide diversity, which was approximately 0.005/bp in all groups. There was some evidence that linkage disequilibrium extended further in northern Europe than in central Europe: the estimates of the population recombination rate parameter, rho, were 0.0064 and 0.0294, respectively. The summary statistics of nucleotide diversity in central and northern European populations were compatible with an ancient bottleneck rather than the standard neutral model.     

Loss of assembly of the main basement membrane collagen, type IV, but not fibril-forming collagens and embryonic death in collagen prolyl 4-hydroxylase I null mice

Collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of the 4-hydroxyproline residues that are essential for the generation of triple helical collagen molecules. The vertebrate C-P4Hs I, II, and III are [alpha(I)]2beta2, [alpha(II)]2beta2, and [alpha(III)]2beta2 tetramers with identical beta subunits. We generated mice with targeted inactivation of the P4ha1 gene encoding the catalytic alpha subunit of C-P4H I to analyze its specific functions. The null mice died after E10.5, showing an overall developmental delay and a dilated endoplasmic reticulum in their cells. The capillary walls were frequently ruptured, but the capillary density remained unchanged. The C-P4H activity level in the null embryos and fibroblasts cultured from them was 20% of that in the wild type, being evidently due to the other two isoenzymes. Collagen IV immunofluorescence was almost absent in the basement membranes of the null embryos, and electron microscopy revealed disrupted basement membranes, while immunoelectron microscopy showed a lack of collagen IV in them. The amount of soluble collagen IV was increased in the null embryos and cultured null fibroblasts, indicating a lack of assembly of collagen IV molecules into insoluble structures, probably due to their underhydroxylation and hence abnormal conformation. In contrast, the null embryos had collagen I and III fibrils with a typical cross-striation pattern but slightly increased diameters, and the null fibroblasts secreted fibril-forming collagens, although less efficiently than wild-type cells. The primary cause of death of the null embryos was thus most likely an abnormal assembly of collagen IV.     

Degraded myelin-associated glycoprotein (dMAG) formation from pure human brain myelin-associated glycoprotein (MAG) is not mediated by calpain or cathepsin L-like activities

The myelin-associated glycoprotein (MAG) is a transmembrane cell adhesion molecule participating in myelin formation and maintenance. Calcium-activated/-dependent proteolysis of myelin-associated glycoprotein by calpain and cathepsin L-like activities has already been detected in purified myelin fractions, producing a soluble fragment, called degraded (d)MAG, characterized by the loss of the transmembrane and cytoplasmic domains. Here, we demonstrate and analyze dMAG formation from pure human brain myelin-associated glycoprotein. The activity never exhibited the high rate previously reported in human myelin fractions. Degradation is time-, temperature-, buffer- and structure-dependent, is inhibited at 4 degrees C and by denaturation of the sample, and is mediated by a trans-acting factor. There is no strict pH dependency of the proteolysis. Degradation was inhibited by excess aprotinin, but not by 1-10 micro g/mL aprotinin and was not eliminated by the use of an aprotinin-sepharose matrix during the purification. dMAG formation was not enhanced by calcium, nor inhibited by a wide variety of protease inhibitors, including specific calpain and cathepsin L inhibitors. Therefore, while cysteine proteases may be present in human myelin membrane fractions, they are not involved in dMAG formation from highly purified human brain myelin-associated glycoprotein preparations.