Novel Method to Load Multiple Genes onto a Mammalian Artificial Chromosome

Mammalian artificial chromosomes are natural chromosome-based vectors that may carry a vast amount of genetic material in terms of both size and number. They are reasonably stable and segregate well in both mitosis and meiosis. A platform artificial chromosome expression system (ACEs) was earlier described with multiple loading sites for a modified lambda-integrase enzyme. It has been shown that this ACEs is suitable for high-level industrial protein production and the treatment of a mouse model for a devastating human disorder, Krabbe’s disease. ACEs-treated mutant mice carrying a therapeutic gene lived more than four times longer than untreated counterparts. This novel gene therapy method is called combined mammalian artificial chromosome-stem cell therapy. At present, this method suffers from the limitation that a new selection marker gene should be present for each therapeutic gene loaded onto the ACEs. Complex diseases require the cooperative action of several genes for treatment, but only a limited number of selection marker genes are available and there is also a risk of serious side-effects caused by the unwanted expression of these marker genes in mammalian cells, organs and organisms. We describe here a novel method to load multiple genes onto the ACEs by using only two selectable marker genes. These markers may be removed from the ACEs before therapeutic application. This novel technology could revolutionize gene therapeutic applications targeting the treatment of complex disorders and cancers. It could also speed up cell therapy by allowing researchers to engineer a chromosome with a predetermined set of genetic factors to differentiate adult stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells into cell types of therapeutic value. It is also a suitable tool for the investigation of complex biochemical pathways in basic science by producing an ACEs with several genes from a signal transduction pathway of interest.     

The contribution of the vegetable material layer to the insulation capacities and water proofing of artificial Mus spicilegus mounds

Successful overwintering of small mammals in temperate and cold climates requires behavioural and physiological adaptations. There are several strategies to survive food shortages and the cold. Most species of small mammals use multiple methods simultaneously but nest building and burrowing are the most widespread among them. A well-constructed, dry nest insulates animals from harsh ambient conditions. Mound-building mice build large banks in the autumn and establish a burrow system with nest chambers beneath them. These overwintering structures are built from soil and a considerable amount of plant material. Recent studies presume that the stored vegetable matter does not, or not exclusively, serve as food and indicate that the mounds might have insulating role. To investigate the function of their plant fill, we built artificial mounds with varying plant content, similar to those built by mound-building mice. We measured temperature change at three levels, at the surface, under the mound and at the nest depth, and investigated their water retaining properties. We showed that the plant fill plays a major role in their thermal insulation and waterproofing properties. Mounds reduced temperature variation of the soil and may protect the nest from absorbing precipitation during the winter.     

Topographic distribution and daily activity of the European ground squirrel population in Bugacpuszta, Hungary

Factors affecting the population density and daily pattern of activity of the European ground squirrelSpermophilus citellus (Linnaeus, 1766) were studied in two habitats of the Kiskunság National Park, Hungary. One habitat was dominated by sandy and the other by salty grassland. Population densities were estimated by counting ground squirrel burrow entrances in sample areas with different microhabitat characteristics. Three categories (elevated, level and depressed) were used to describe the spatial distribution of burrows according to microtopography. Daily activity was evaluated by counting the ground squirrels visible on the surface and registering their alarm calls. The highest proportion of ground squirrel holes was found on elevations in the two habitats. Ground squirrels showed a similar daily activity pattern in different areas and in the same season in consecutive years. The daily activity consisted in a morning peak, a midday pause and a less regular afternoon activity period. Human activity had no direct effect on the daily activity pattern. Emission of alarm calls peaked in June, when litters emerge from the maternal burrows.     

Expression of caveolin‐1 in the interfollicular but not the follicle‐associated epithelial cells in the bursa of fabricius of chickens

The surface epithelium of the bursa of Fabricius consists of interfollicular (IFE) and follicle‐associated epithelium (FAE). The IFE comprises (i) cylindrical‐shaped secretory cells (SC) and (ii) cuboidal basal cells (BCs). The FAE provides histological and two‐way functional connections between the bursal lumen and medulla of the follicle. We used a carbon solution and anti‐caveolin‐1 (Cav‐1) to study the endocytic activity of FAE. Carbon particles entered the intercellular space of FAE, but the carbon particles were not internalized by the FAE cells. Cav‐1 was not detectable in the FAE cells or the medulla of the bursal follicle. The absence of Cav‐1 indicates that no caveolin‐mediated endocytosis occurs in the FAE cells, B cells, bursal secretory dendritic cells (BSDC), or reticular epithelial cells. Surprisingly, a significant number of Cav‐1 positive cells can be found among the SC, which are designated SC II. Cav‐1 negative cell are called SC I, and they produce mucin for lubricating the bursal lumen and duct. Occasionally, BCs also express Cav‐1, which suggests that BC is a precursor of a SC. Transmission electron microscopy confirmed the existence of type I and II SC. The SC II are highly polarized and have an extensive trans‐Golgi network that is rich in different granules and vesicles. Western blot analysis of bursa lysates revealed a 21–23 kDa compound (caveolin) and Filipin fluorescence histochemistry provided evidence for intracellular cholesterol. High amount of cholesterol in the feces shows the cholesterol efflux from SC II. The presence of Cav‐1 and cholesterol in SC II indicates, that the bursa is a complex organ in addition to possessing immunological function contributes to the cholesterol homeostasis in the chickens.     

Diuretic treatment affects the length of torpor bouts in hibernating European ground squirrels (Spermophilus citellus)

During the hibernation season, hibernating mammals show a sequence of torpor bouts that are interrupted by periodic arousals and brief normothermic periods. The functional significance of periodic arousals is still uncertain. We hypothesized that the imbalances in water economy may play a role in the timing of periodic arousals in hibernating species. We applied furosemide, a diuretic drug, to assess whether hibernating European ground squirrels respond to elevated urine production by shortening their torpor bouts. Urine production in the treated squirrels increased and led to more frequent arousals, presumably to restore water balance by recovery of lost water from blood and tissues. The length of the subsequent normothermic phase was not affected by the diuretic treatment. Body mass change correlated primarily with the amount of voided urine. Although our study did not identify the underlying mechanism, our results support the view that water economy, and water loss may play a role in the timing of periodic arousals.     

Polymorphism and inheritance of serum esterases and β-globulins in the paradise fish (Macropodus opercularis; Anabantidae)

Electrophoretic variants of serum esterases and β-globulins in two subspecies of paradise fish ( Macropodus opercularis ) were studied. Four esterase loci ( Est-1, Est-2, Est-3 and Est-4 ), a single transferin ( Tf ) and another major β-globulin locus ( Bg ) were identified by segregational analysis. Est-3 seems to be a monomorphic. locus. Three alleles of Est-1 , two of Est-2 , two of Est-4 , four of Tf and two alleles of Bg were found in the laboratory population. None of these loci were closely linked. Electrophoretic patterns of F₁ hybrids confirmed the monomeric structures of each of the studied proteins. Allelic segregation at the Tf and Bg loci was normal in F₂ and backcross populations. In crosses of the two Macropodus subspecies there were deviations from Mendelian ratios because of missing recombinant esterase phenotypes. Each of these would have been homozygous Est-2^f/f . We suppose that Est-2^f/f causes lethality in the early phase of development, except in the Est-1^c/c, Est-2^f/f combination characteristic of the parental subspecies M.o. concolor .     

Successful Application of Video-Projected Human Images for Signalling to Dogs

Dogs were tested (1) in a two‐way choice experiment, where the experimenter indicated a baited bowl by pointing; and (2) in a task where the owner was asked to command the dog to execute simple obedience tasks. In expt 1 dogs (n = 10) were tested at first in the presence of the experimenter (three dimensional condition, 3D), that was followed by a series of pointing trials when the life‐sized image of the experimenter was projected onto the wall by the means of a video‐projector (two dimensional condition, 2D). Dogs performed correctly more often than expected by chance in both 3D and 2D conditions. In expt 2 the commanding owner was either present in the room (3D), or her/his image was projected on the screen (2D), or only her/his voice was audible for the dog through a speaker (0D). The performance of the dogs (n = 10) decreased to great extent comparing the 3D and 0D condition, as the number of different actions the dogs obeyed was significantly less in the 0D condition. However, there was no difference in the number of different actions obeyed in the 3D and 2D conditions. Our results show that dogs readily obey life‐sized, interactive moving image in various communicative situtations. We suppose that the difference between 2D and 3D conditions can be attributed partially to the lack of some additional communicative signals as sounds (verbal cues) and odours (from the human), and to some changes in the context.     

Novel perspectives of target-binding by the evolutionarily conserved PP4 phosphatase

Protein phosphatase 4 (PP4) is an evolutionarily conserved and essential Ser/Thr phosphatase that regulates cell division, development and DNA repair in eukaryotes. The major form of PP4, present from yeast to human, is the PP4c-R2-R3 heterotrimeric complex. The R3 subunit is responsible for substrate-recognition via its EVH1 domain. In typical EVH1 domains, conserved phenylalanine, tyrosine and tryptophan residues form the specific recognition site for their target''s proline-rich sequences. Here, we identify novel binding partners of the EVH1 domain of the Drosophila R3 subunit, Falafel, and demonstrate that instead of binding to proline-rich sequences this EVH1 variant specifically recognizes atypical ligands, namely the FxxP and MxPP short linear consensus motifs. This interaction is dependent on an exclusively conserved leucine that replaces the phenylalanine invariant of all canonical EVH1 domains. We propose that the EVH1 domain of PP4 represents a new class of the EVH1 family that can accommodate low proline content sequences, such as the FxxP motif. Finally, our data implicate the conserved Smk-1 domain of Falafel in target-binding. These findings greatly enhance our understanding of the substrate-recognition mechanisms and function of PP4.