Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: A useful model to monitor germ cells in a live vertebrate

We have generated transgenic medaka (teleost, Oryzias latipes), which allow us to monitor germ cells by green fluorescent protein (GFP) fluorescence in live specimens. Two medaka strains, himedaka (orange–red variety) and inbred QurtE, were used. The transgenic lines were achieved by microinjection of a construct containing the putative promoter region and 3′ region of the medaka vasa gene (olvas). The intensity of GFP fluorescence increases dramatically in primordial germ cells (PGCs) located in the ventrolateral region of the posterior intestine around stage 25 (the onset of blood circulation). Whole-mount in situ hybridization and monitoring of ectopically located cells by GFP fluorescence suggested that (i) the increase in zygotic olvas expression occurs after PGC specification and (ii) PGCs can maintain their cell characteristics ectopically after stages 20–25. Around the day of hatching, the QurtE strain clearly exhibits sexual dimorphisms in the number of GFP fluorescent germ cells, a finding consistent with the appearance of leucophores, a sex-specific marker of QurtE. The GFP expression persists throughout the later stages in the mature ovary and testis. Thus, these transgenic medaka represent a live vertebrate model to investigate how germ cells migrate to form sexually dimorphic gonads, as well as a potential assay system for environmental substances that may affect gonad development. The use of a transgenic construct as a selective marker to efficiently isolate germ-line-transmitting founders during embryogenesis is also discussed.

Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na+/K+ translocating AtHKT1 protein, a member of the superfamily of K+ transporters

The Arabidopsis thaliana AtHKT1 protein, a Na+/K+ transporter, is capable of mediating inward Na+ currents in Xenopus laevis oocytes and K+ uptake in Escherichia coli. HKT1 proteins are members of a superfamily of K+ transporters. These proteins have been proposed to contain eight transmembrane segments and four pore-forming regions arranged in a mode similar to that of a K+ channel tetramer. However, computer analysis of the AtHKT1 sequence identified eleven potential transmembrane segments. We have investigated the membrane topology of AtHKT1 with three different techniques. First, a gene fusion alkaline phosphatase study in E. coli clearly defined the topology of the N-terminal and middle region of AtHKT1, but the model for membrane folding of the C-terminal region had to be refined. Second, with a reticulocyte-lysate supplemented with dog-pancreas microsomes, we demonstrated that N-glycosylation occurs at position 429 of AtHKT1. An engineered unglycosylated protein variant, N429Q, mediated Na+ currents in X. laevis oocytes with the same characteristics as the wild-type protein, indicating that N-glycosylation is not essential for the functional expression and membrane targeting of AtHKT1. Five potential glycosylation sites were introduced into the N429Q. Their pattern of glycosylation supported the model based on the E. coli-alkaline phosphatase data. Third, immunocytochemical experiments with FLAG-tagged AtHKT1 in HEK293 cells revealed that the N and C termini of AtHKT1, and the regions containing residues 135–142 and 377–384, face the cytosol, whereas the region of residues 55–62 is exposed to the outside. Taken together, our results show that AtHKT1 contains eight transmembrane-spanning segments.

Photoactive yellow protein: A structural prototype for the three-dimensional fold of the PAS domain superfamily

PAS domains are found in diverse proteins throughout all three kingdoms of life, where they apparently function in sensing and signal transduction. Although a wealth of useful sequence and functional information has become recently available, these data have not been integrated into a three-dimensional (3D) framework. The very early evolutionary development and diverse functions of PAS domains have made sequence analysis and modeling of this protein superfamily challenging. Limited sequence similarities between the ∼50-residue PAS repeats and one region of the bacterial blue-light photosensor photoactive yellow protein (PYP), for which ground-state and light-activated crystallographic structures have been determined to high resolution, originally were identified in sequence searches using consensus sequence probes from PAS-containing proteins. Here, we found that by changing a few residues particular to PYP function, the modified PYP sequence probe also could select PAS protein sequences. By mapping a typical ∼150-residue PAS domain sequence onto the entire crystallographic structure of PYP, we show that the PAS sequence similarities and differences are consistent with a shared 3D fold (the PAS/PYP module) with obvious potential for a ligand-binding cavity. Thus, PYP appears to prototypically exhibit all the major structural and functional features characteristic of the PAS domain superfamily: the shared PAS/PYP modular domain fold of ∼125–150 residues, a sensor function often linked to ligand or cofactor (chromophore) binding, and signal transduction capability governed by heterodimeric assembly (to the downstream partner of PYP). This 3D PAS/PYP module provides a structural model to guide experimental testing of hypotheses regarding ligand-binding, dimerization, and signal transduction.

Nucleotide sequences provide evidence of genetic exchange among distantly related lineages of Trypanosoma cruzi

Simple phylogenetic tests were applied to a large data set of nucleotide sequences from two nuclear genes and a region of the mitochondrial genome of Trypanosoma cruzi, the agent of Chagas' disease. Incongruent gene genealogies manifest genetic exchange among distantly related lineages of T. cruzi. Two widely distributed isoenzyme types of T. cruzi are hybrids, their genetic composition being the likely result of genetic exchange between two distantly related lineages. The data show that the reference strain for the T. cruzi genome project (CL Brener) is a hybrid. Well-supported gene genealogies show that mitochondrial and nuclear gene sequences from T. cruzi cluster, respectively, in three or four distinct clades that do not fully correspond to the two previously defined major lineages of T. cruzi. There is clear genetic differentiation among the major groups of sequences, but genetic diversity within each major group is low. We estimate that the major extant lineages of T. cruzi have diverged during the Miocene or early Pliocene (3–16 million years ago).

Higher Activity of an Aldehyde Oxidase in the Auxin-Overproducing superroot1 Mutant of Arabidopsis thaliana1

Aldehyde oxidase (AO; EC 1.2.3.1) activity was measured in seedlings of wild type or an auxin-overproducing mutant, superroot1 (sur1), of Arabidopsis thaliana. Activity staining for AO after native polyacrylamide gel electrophoresis separation of seedling extracts revealed that there were three major bands with AO activity (AO1–3) in wild-type and mutant seedlings. One of them (AO1) had a higher substrate preference for indole-3-aldehyde. This AO activity was significantly higher in sur1 mutant seedlings than in the wild type. The difference in activity was most apparent 7 d after germination, the same time required for the appearance of the remarkable sur1 phenotype, which includes epinastic cotyledons, elongated hypocotyls, and enhanced root development. Higher activity was observed in the root and hypocotyl region of the mutant seedlings. We also assayed the indole-3-acetaldehyde oxidase activity in extracts by high-performance liquid chromatography detection of indole-3-acetic acid (IAA). The activity was about 5 times higher in the extract of the sur1 seedlings, indicating that AO1 also has a substrate preference for abscisic aldehyde. Treatment of the wild-type seedlings with picloram or IAA caused no significant increase in AO1 activity. This result suggested that the higher activity of AO1 in sur1 mutant seedlings was not induced by IAA accumulation and, thus, strongly supports the possible role of AO1 in IAA biosynthesis in Arabidopsis seedlings.

Excitatory versus inhibitory GABA as a divergence point in steroid-mediated sexual differentiation of the brain

Whereas adult sex differences in brain morphology and behavior result from developmental exposure to steroid hormones, the mechanism by which steroids differentiate the brain is unknown. Studies to date have described subtle sex differences in levels of proteins and neurotransmitters during brain development, but these have lacked explanatory power for the profound sex differences induced by steroids. We report here a major divergence in the response to injection of the γ-aminobutyric acid type A (GABAA) agonist, muscimol, in newborn male and female rats. In females, muscimol treatment primarily decreased the phosphorylation of cAMP response element binding protein (CREB) within the hypothalamus and the CA1 region of the hippocampus. In contrast, muscimol increased the phosphorylation of CREB in males within these same brain regions. Within the arcuate nucleus, muscimol treatment increased the phosphorylation of CREB in both females and males. Thus, the response to GABA can be excitatory or inhibitory on signal-transduction pathways that alter CREB phosphorylation depending on the sex and the region in developing brain. This divergence in response to GABA allows for a previously unknown form of steroid-mediated neuronal plasticity and may be an initial step in establishing sexually dimorphic signal-transduction pathways in developing brain.

Mapping the protein regions responsible for the functional specificities of the Arabidopsis MADS domain organ-identity proteins.

The Arabidopsis MADS domain proteins AP1, AP3, PI, and AG specify floral organ identity. All of these proteins contain a MADS domain required for DNA binding and dimerization; a region termed L (linker between MADS domain and K domain), which plays an important role in dimerization specificity; the K domain, named for its similarity to the coiled-coil domain of keratin; and a C-terminal region of unknown function. To determine which regions of these proteins are responsible for their abilities to specify different organs, we have made a number of chimeric MADS box genes. The in vivo function of these chimeric genes was investigated by ectopic expression in transgenic Arabidopsis plants. The four proteins fall into two classes on the basis of regions responsible for their functional specificities. The L region and K domain define the functional specificities of AP3 and PI, while the MADS domain and L region define the functional specificities of AP1 and AG.

Review of the Economic Development in the Western Balkan States. OSW STUDIES No. 19, 2005-08-10

Foreword. Ten years after the end of the armed conflict, the Western Balkans1 are still being considered as the “land of the unsuccessful policies”. Enormous financial and technical assistance transferred by the International Community has not managed to meet the goals of integrating the region within itself as well as within the European markets. Explanation for this can be found in the consequences of the war and the remnants of the socialist state. The complexity of current institutional/ political arrangements combined with the limited willingness of the regional actors to introduce and implement much of the needed reforms have additionally contributed to the current state of affairs. The economy and politics in the region intertwine to an extent as probably in none of the other post-communist states. Therefore, the paper presents the recent economic performance of the Western Balkan countries in the light of their limited institutional development and lack of efficient regional cooperation. The paper discusses the importance of foreign direct investments’ inflow for the economic growth of the “latecomer” states and presents major drawbacks which limit the influx of the foreign capital to the region. It presents private sector activity and regional cooperation programmes. It discusses the role of the International Community with the main focus on the activities of the European Union. The EU is examined not only as the main aid donor but more importantly as a foreign trade partner. Furthermore, it analyses the impact of the presence of the International Community and their strategies towards the region with the special attention to the EU. Finally, it presents recommendations for the improvement of the economic performance in light of the enhanced political cooperation between the EU and the region.

The Baltic states’ Territorial Defence Forces in the face of hybrid threats. OSW Commentary Number 165, 19.03.2015

From the Introduction. Russia’s annexation of Crimea and its aggression in eastern Ukraine have triggered a debate about the main directions of defence policy in the Nordic and Baltic region. In the Baltic states, but also in the Nordic countries and Poland, much attention is being paid to questions of Territorial Defence Forces (TDF). TDF are viewed as one of the elements in the national defence systems’ response during the early stages of a hybrid conflict. The Baltic states have decided to adapt their Territorial Defence Forces to new threats by making a number of changes to their functioning, depending on the local conditions in each case. Given the growing uncertainty in the region, they have opted not to undertake any in-depth reforms of TDF at this stage, as that could entail a temporary disorganisation in the armed forces. In the coming years Estonia, Latvia and Lithuania will invest in increasing the size and combat readiness of their Territorial Defence Forces, providing them with better training and equipment, and creating a system of incentives to encourage more people to serve in volunteer formations.

An insight into the breakup of Gondwana: Identifying events through low-temperature thermochronology from the basement rocks of Madagascar

Fission track analysis was applied to the Precambrian suites of Madagascar in order to identify the lower-temperature cooling histories and their relationships to the Phanerozoic events that affected the island. Apatite ages range from 431 to 68 Ma, and zircon ages range from 452 to 238 Ma. Thermochronologically, the island can be divided into a southern, central, and northern region each with a subdivision on an east-west basis. The southern region is sharply separated from the central region by strongly contrasting apparent apatite ages over the northwest-southeast striking Ranotsara Shear Zone (RSZ). The change in apparent ages over the RSZ is indicative of later reactivation along younger brittle faults. The central region has the oldest ages of the island and has a diffuse contact to the third region northward. Along the entire western margin of the Precambrian basement initial Paleozoic exhumation was followed by heating (burial by sediments) during Jurassic and Cretaceous times. A decrease in ages along the eastern margin from 119 to 68 Ma coincides with the predicted positions of the Marion hot spot after effects of erosion are considered. On the other hand, these ages may represent progressive opening of the margin in a southward direction together with associated denudation of the rift shoulder. The eastern part of the central region has remained very stable since at least Devonian times, undergoing only long-term very slow exhumation at rates of 1–5 m/Myr.

Sedimentology of the Rio Grande Rise area

Surface sediment samples and three gravity cores from the eastern terrace of the Vema Channel, the western flank of the Rio Grande Rise, and the Brazilian continental slope were investigated for physical properties, grain size, and clay mineral composition. Discharge of the Rio Doce is responsible for kaolinite enrichments on the slope south of 20° and at intermediate depths of the Rio Grande Rise. The long-distance advection of kaolinite with North Atlantic Deep Water from lower latitudes is of minor importance as evidenced by low kaolinite/chlorite ratios on the Mid-Atlantic Ridge. Cyclic variations of kaolinite/chlorite ratios in all our cores, with maxima in interglacials, are attributed to low-and high-latitude forcing of paleoclimate on the Brazilian mainland and the related discharge of the Rio Doce. A long-term trend toward more arid and 'glacial' conditions from 1500 ka to present is superimposed on the glacial-interglacial cyclicity.

Table 1: Main characteristics of the BIOTA observatories included in this study & Table 3: Occurrence of termite taxa at the observatories

Termites are the most important soil ecosystem engineers of semi-arid and arid habitats. They enhance decomposition processes as well as the subsequent mineralisation of nutrients by bacteria and fungi. Through their construction of galleries, nests and mounds, they promote soil turnover and influence the distribution of nutrients and also alter texture and hydrological properties of soils, thereby affecting the heterogeneity of their ecosystem. The main aim of the present thesis was to define the impact of termites on ecosys-tem functioning in a semi-arid ecosystem. In a baseline study, I assessed the diversity of termite taxa in relation to the amount of precipitation, the vegetation patterns and the land use systems at several sites in Namibia. Subsequently, I focussed on a species that is highly abundant in many African savannas, the fungus growing and mound building species Macro-termes michaelseni (Sjöstedt, 1914). I asked how this species influences the spatial hetero-geneity of soil and vegetation patterns. From repeated samplings at 13 sites in Namibia, I obtained 17 termite taxa of 15 genera. While the type of land use seems to have a minor effect on the termite fauna, the mean annual precipitation explained 96% and the Simpson index of vascular plant diversity 81% of the variation in taxa diversity. The number of termite taxa increased with both of these explanation variables. In contrast to former studies on Macrotermes mounds in several regions of Africa that I reviewed, soil analyses from M. michaelseni mounds in the central Namibian savanna revealed that they contain much higher nitrogen contents when compared to their parent material. Further analyses revealed that nitrate forms a major component of the nitrogen content in termite mounds. As nitrate solves easily in water, evaporation processes are most probably responsible for the transport of solved nitrates to the mound surface and their accumulation there. The analysed mounds in central Namibia contained higher sand propor-tions compared to the mounds of the former studies. Through the higher percentage of coarse and middle sized pores, water moves more easily in sandy soils compared to more clayey soils. In consequence, evaporation-driven nitrate accumulation can occur in the studied mounds at high rates. ff...

Project Aurora III; final report. Contract no. AF 19(628)-380, Project no. 5631, Task no. 563111. Period covered: 12 February 1962 through 15 June 1965.

"AFCRL-65-451"

Beyond the Bauhaus : Cultural Modernity in Breslau, 1918-1933 /

The Breslau arts scene during the Weimar period was one of the most vibrant in all of Germany, yet it has disappeared from memory and historiography. Breslau was a key center for innovative artistic production during the Weimar Republic; recovery of its history will shed new light on German cultural dynamics in the 1920s. Such a study has art historical significance because of the incredible extent of innovation that occurred in almost every intellectual field, advances that formed the basis for aesthetic modernism internationally and continue to affect the course of visual art and architecture today. Architecture education, just one example in many, is still largely based on a combination of the Bauhaus model from the 1920s and the model developed at the Breslau Academy of Fine and Applied Art. The exploratory attitude encouraged in Weimar era arts endeavors, as opposed to the conformism of academic art, is still a core value promoted in contemporary art and architecture circles. Given the long-lasting influence of Weimar culture on modernism one would expect to find a spate of studies examining every aspect of its cultural production, but this is not the case. Recent scholarship is almost exclusively focused on Berlin and the Dessau Bauhaus. Although both interests are understandable, the creative explosion was not confined to these cities but was part of a larger cultural ethos that extended into many of the smaller regional centers. The Expressionist associations the Blaue Reiter in Munich and Brücke in Dresden are two well-known examples. Equally, innovation was not confined to a few monumental projects like the Stuttgart Weissenhofsiedlung but part of a broader national cultural ethos. The dispersion of modernism occurred partly because of the political history of Germany as a loosely joined confederation of small city states and principalities that had strong individual cultural identities before unification in 1871 but also because of the German propensity to value and take intense pride in the Heimat, understood both as the hometown and the region. Heimatliebe translated into generous support for cultural institutions in outlying cities. Host to a roster of internationally acclaimed artists and architects, major collectors, arts organizations, museums, presses, galleries, and one of the premier German arts academies of the day, Breslau boasted a thriving modern arts scene until 1933 when the Nazis began their assault on so-called "degenerate" art. This book charts the cultural production of Breslau-based artists, architects, art collectors, urban designers, and arts educators, who were especially interesting because they operated in the space between the margins of Weimar-era cultural debates. Rather than accepting the radical position of the German avant-garde or the reactionary position of German conservatives, many Breslauers sought a middle ground. It is the first book in English to address this history and presents the history in a manner unique to any studies currently on the market. 'Beyond the Bauhaus' explores the polyvalent and contradictory nature of cultural production in Breslau in order to expand the cultural and geographic scope of Weimar history; the book asserts a reciprocal dimension to the relationship between regional culture and national culture, between centers like Breslau and the capital Berlin. With major international figures like the painters Otto Mueller and Oskar Moll, architects Hans Scharoun and Adolf Rading, urban planners Max Berg and Ernst May, collectors Ismar Littmann and Max Silberberg, and an art academy that by 1929 was considered the best in Germany, Breslau clearly had significance to narratives of Weimar cultural production. 'Beyond the Bauhaus' contributes the history of German culture during the Weimar Republic. It belongs alongside histories of art, architecture, urban design, exhibition, collecting, and culture; histories of the Bauhaus; histories of arts education more broadly; and German history. The readership would include those interested in German history; German art, architecture, urban design, planning, collecting, and exhibition history; in the avant-garde; the development of arts academies and arts pedagogy; and the history of Breslau and Silesia.

Raven management plan for the California Desert Conservation Area : draft /

In cooperation with: California Dept. of Fish and Game, Region IV and Region V, and U.S. Fish and Wildlife Service.