Intermediate filament genes as differentiation markers in the leech <Emphasis Type="Italic">Helobdella</Emphasis>

The intermediate filament (IF) cytoskeleton is a general feature of differentiated cells. Its molecular components, IF proteins, constitute a large family including the evolutionarily conserved nuclear lamins and the more diverse collection of cytoplasmic intermediate filament (CIF) proteins. In vertebrates, genes encoding CIFs exhibit cell/tissue type-specific expression profiles and are thus useful as differentiation markers. The expression of invertebrate CIFs, however, is not well documented. Here, we report a whole-genome survey of IF genes and their developmental expression patterns in the leech Helobdella, a lophotrochozoan model for developmental biology research. We found that, as in vertebrates, each of the leech CIF genes is expressed in a specific set of cell/tissue types. This allows us to detect earliest points of differentiation for multiple cell types in leech development and to use CIFs as molecular markers for studying cell fate specification in leech embryos. In addition, to determine the feasibility of using CIFs as universal metazoan differentiation markers, we examined phylogenetic relationships of IF genes from various species. Our results suggest that CIFs, and thus their cell/tissue-specific expression patterns, have expanded several times independently during metazoan evolution. Moreover, comparing the expression patterns of CIF orthologs between two leech species suggests that rapid evolutionary changes in the cell or tissue specificity of CIFs have occurred among leeches. Hence, CIFs are not suitable for identifying cell or tissue homology except among very closely related species, but they are nevertheless useful species-specific differentiation markers.     

Structure,composition and mechanical properties of the silk fibres of the egg case of the Joro spider, <Emphasis Type="Italic">Nephila clavata</Emphasis> (Araneae,Nephilidae)

The silk egg case and orb web of spiders are elaborate structures that are assembled from a number of components. We analysed the structure, the amino acid and fibre compositions, and the tensile properties of the silk fibres of the egg case of Nephila clavata. SEM shows that the outer and inner covers of the egg case consist of thick, medium and thin silk fibres. The silk fibres of the outer cover of the egg case are probably produced by the major and minor ampullate glands. The silk fibres of the inner cover of the egg case from cylindrical glands appears to be distinct from the silk fibres of the major ampullate glands based on their micro-morphology, mole percent amino acid composition and types, and tensile behaviour and properties. Collectively, our investigations show that N. clavata uses silk fibres from relatively few glands in varying combinations to achieve different physical and chemical properties (e.g., color, diameter, morphology and amino acid composition) and functional and mechanical properties in the different layers of the egg case.     

Comparative analysis of the informativeness of ISSR markers for estimating genetic diversity of horse breeds

The informativeness of six different types of ISSR markers was compared in order to evaluate genetic diversity of Mongolian, Buryat and Tuvinian horse breeds (Equus caballus). The dinucleotide-based ISSR primers ((GA)₉C, (AG)₉C, and (CA)₉G) proved to be of little use to examine genetic polymorphism among horses due to the low number of amplified and polymorphic fragments. The polymorphism information content (PIC) for the dinucleotide-based ISSR primers was 2.88 times lower than that for trinucleotide-based ISSR markers. Among the trinucleotide-based ISSR markers, CAC ISSR marker was also found to be noninformative, due to the low number of polymorphic loci identified. The highest values of the polymorphism information content (PIC) were obtained for two types of ISSR markers, based on (GAG)₆C (PIC, 3.61) and on (ACC)₆G (PIC, 2.50). Thus, the results of the present study showed that GAG and ACC ISSR markers were most informative for evaluation of genetic diversity among horses.     

Surface Water Linkages Regulate Trophic Interactions in a Groundwater Food Web

Groundwaters are increasingly viewed as resource-limited ecosystems in which fluxes of dissolved organic carbon (DOC) from surface water are efficiently mineralized by a consortium of microorganisms which are grazed by invertebrates. We tested for the effect of groundwater recharge on resource supply and trophic interactions by measuring physico-chemistry, microbial activity and biomass, structure of bacterial communities and invertebrate density at three sites intensively recharged with surface water. Comparison of measurements made in recharge and control well clusters at each site showed that groundwater recharge significantly increased fluxes of DOC and phosphate, elevated groundwater temperature, and diminished dissolved oxygen (DO). Microbial biomass and activity were significantly higher in recharge well clusters but stimulation of autochthonous microorganisms was not associated with a major shift in bacterial community structure. Invertebrate assemblages were not significantly more abundant in recharge well clusters and did not show any relationship with microbial biomass and activity. Microbial communities were bottom-up regulated by DOC and nutrient fluxes but trophic interactions between microorganisms and invertebrates were apparently limited by environmental stresses, particularly DO depletion and groundwater warming. Hydrological connectivity is a key factor regulating the function of DOC-based groundwater food webs as it influences both resource availability for microorganisms and environmental stresses which affect energy transfer to invertebrates and top-down control on microorganisms.     

Intergenerational effect of juvenile hormone on offspring in <Emphasis Type="Italic">Pogonomyrmex</Emphasis> harvester ants

Parents can influence the phenotypes of their offspring via a number of mechanisms. In harvester ants, whether female progeny develop into workers or daughter queens is strongly influenced by the age and temperature conditions experienced by their mother, which is associated with variation in maternal ecdysteroid deposition in fertilized eggs. In many insects, juvenile hormone (JH) is antagonistic to ecdysteroid release, suggesting that seasonal and age-based variation in maternal JH titers may explain maternal effects on offspring size and reproductive caste. To test this hypothesis, we artificially increased maternal JH titers with methoprene, a JH analog, in laboratory colonies of two Pogonomyrmex populations exhibiting genetic caste determination. Increasing maternal JH resulted in a 50% increase in worker body size, as well as a sharp reduction in total number of progeny reared, but did not alter the genotype of progeny reared to adulthood. The intergenerational effect of JH manipulation was not mediated by a reduction in ecdysteroid deposition into eggs; instead, changes in egg size, trophic egg availability or brood/worker ratio may have altered the nutritional environment of developing larvae. Egg ecdysteroid content was significantly negatively correlated with natural variation in worker body size, however, suggesting that there are multiple independent routes by which queens can modify offspring phenotypes.     

Defoliation depletes the carbohydrate reserves of resprouting <Emphasis Type="Italic">Acacia</Emphasis> saplings in an African savanna

Over the past century there has been a global trend towards tree expansion and densification in rangelands and savannas. This phenomenon is commonly referred to as bush encroachment. In South Africa Acacia karroo is one of the key species responsible for bush encroachment. It has been suggested that the combination of fire and browsing might limit bush encroachment by A. karroo more effectively than either browsing or fire alone. We hypothesized that these repeated disturbances progressively deplete root carbohydrates and compromise resprouting ability. This was tested by burning and then manually defoliating A. karroo once a month for 1 year. Manual defoliation did not inhibit the rapid shoot elongation after topkill of A. karroo saplings. During this initial phase, the growth of the new shoots of A. karroo was dependent more on mobilised root reserves than on photoassimilates from the new shoots. Frequent manual defoliation of resprouting A. karroo saplings prevented the replenishment of starch reserves. We suggest a mechanism for how the interaction of browsing and fire can suppress and perhaps reverse bush encroachment in African savannas. Saplings that have reduced starch reserves at the end of dry season due to browsing will struggle to resprout if they are burnt. Even if they do not die, they will be less able to escape fire damage in the next fire than if they had been able to resprout unimpeded.     

Summer zooplankton in small rivers in relation to selected conditions

The abundance and the biodiversity of summer zooplankton in the waters of the Drawa drainage (NW Poland) were studied, as was their relation to selected environmental conditions. The conditions upstream, especially in the outlets of lakes, did not affect the zooplankton communities downstream. This was also true of tributaries which had no influence on the shape of the zooplankton communities in the main river. The number of zooplankton in the outlets of eutrophic lakes was greater than in those of mesotrophic lakes. Increased vegetation cover significantly affected mainly the crustacean communities of zooplankton. Larger amounts of zooplankton were observed in rivers where the riparian zone was not covered with vegetation, but this difference was not significant. The hydrological conditions of the rivers and the Secchi depth visibility strongly impacted the composition of the zooplankton. The influence of abiotic factors was most pronounced on the abundance of cladocerans, and least pronounced on the abundance of rotifers.     

The Mental Retardation Associated Protein,srGAP3 Negatively Regulates VPA-Induced Neuronal Differentiation of Neuro2A Cells

The Slit-Robo GTPase-activating proteins (srGAPs) are important multifunctional adaptor proteins involved in various aspects of neuronal development, including axon guidance, neuronal migration, neurite outgrowth, dendritic morphology and synaptic plasticity. Among them, srGAP3, also named MEGAP (Mental disorder-associated GTPase-activating protein), plays a putative role in severe mental retardation. SrGAP3 expression in ventricular zones of neurogenesis indicates its involvement in early stage of neuronal development and differentiation. Here, we show that overexpression of srGAP3 inhibits VPA (valproic acid)-induced neurite initiation and neuronal differentiation in Neuro2A neuroblastoma cells, whereas knockdown of srGAP3 facilitates the neuronal differentiation in this cell line. In contrast to the wild type, overexpression of srGAP3 harboring an artificially mutation R542A within the functionally important RhoGAP domain does not exert a visible inhibitory effect on neuronal differentiation. The endogenous srGAP3 selectively binds to activated form of Rac1 in a RhoGAP pull-down assay. We also show that constitutively active (CA) Rac1 can rescue the effect of srGAP3 on attenuating neuronal differentiation. Furthermore, change in expression and localization of endogenous srGAP3 is observed in neuronal differentiated Neuro2A cells. Together, our data suggest that srGAP3 could regulate neuronal differentiation in a Rac1-dependent manner.     

Non-erythroid beta spectrin interacting proteins and their effects on spectrin tetramerization

With yeast two-hybrid methods, we used a C-terminal fragment (residues 1697–2145) of non-erythroid beta spectrin (βII-C), including the region involved in the association with alpha spectrin to form tetramers, as the bait to screen a human brain cDNA library to identify proteins interacting with βII-C. We applied stringent selection steps to eliminate false positives and identified 17 proteins that interacted with βII-C (IP_βII-C s). The proteins include a fragment (residues 38–284) of “THAP domain containing, apoptosis associated protein 3, isoform CRA g”, “glioma tumor suppressor candidate region gene 2” (residues 1-478), a fragment (residues 74–442) of septin 8 isoform c, a fragment (residues 704–953) of “coatomer protein complex, subunit beta 1, a fragment (residues 146–614) of zinc-finger protein 251, and a fragment (residues 284–435) of syntaxin binding protein 1. We used yeast three-hybrid system to determine the effects of these βII-C interacting proteins as well as of 7 proteins previously identified to interact with the tetramerization region of non-erythroid alpha spectrin (IP_αII-N s) [1] on spectrin tetramer formation. The results showed that 3 IP_βII-C s were able to bind βII-C even in the presence of αII-N, and 4 IP_αII-N s were able to bind αII-N in the presence of βII-C. We also found that the syntaxin binding protein 1 fragment abolished αII-N and βII-C interaction, suggesting that this protein may inhibit or regulate non-erythroid spectrin tetramer formation.     

Characterization and pharmacological properties of in vitro propagated clones of <Emphasis Type="Italic">Echinacea tennesseensis</Emphasis> (Beadle) Small

Tissue culture techniques have been used to establish and maintain a repository of medicinal Echinacea. In vitro clones obtained from hypocotyls of germinated seeds, varied macroscopically, microscopically and exhibited variation in immune enhancing activity. Two in vitro produced clones of Echinacea tennesseensis (Beadle) Small (ETN 03 and ETN 11) were identified as high and low activity based on the activation of human monocytes. Phenotypic analyses of ETN 03 and ETN 11 clones were done using AFLP (Amplified Fragment Length Polymorphism) assay. Results of the AFLP assay revealed that no mutation has occurred during in vitro multiplication, storage, and acclimatization into soil. Plants of ETN 03, ETN 11 clones were cultivated for two growing seasons. Extracts of their dry leaves and roots exhibited immune enhancing activity; however, the variation in activity noticed between clones during micropropagation diminished and was no longer statistically relevant.