Short Inverted-Repeat Transposable Elements in Teleost Fish and Implications for a Mechanism of Their Amplification

Angel is the first miniature inverted-repeat transposable element (MITE) isolated from fish. Angel elements are imperfect palindromes with the potential to form stem-loop structures in vitro. Despite sequence divergence of elements of up to 55% within and between species, their inverted repeat structures have been maintained, implying functional importance. We estimate that there are about 10³–10⁴ Angels scattered throughout the zebrafish genome, evidence that this family of transposable elements has been significantly amplified over the course of evolution. Angel elements and Xenopus MITEs carry common sequence motifs at their termini, indicating common origin and/or related mechanisms of transposition. We present a model in which MITEs take advantage of the basic cellular mechanism of DNA replication for their amplification, which is dependent on the characteristic inverted repeat structures of these elements. We propose that MITEs are genomic parasites that transpose via a DNA intermediate, which forms by a folding-back of a single strand of DNA, that borrow all of the necessary factors for their amplification from products encoded in the genomes in which they reside. DNA polymorphisms in different lines of zebrafish were detected by PCR using Angel-specific primers, indicating that such elements, combined with other transposons in vertebrate genomes, will be useful molecular tools for genome mapping and genetic analyses of mutations. Received: 7 April 1998 / Accepted: 7 April 1998     

Long-term steady-state labelling of wheat plants by use of natural 13CO2/12CO2 mixtures in an open,rapidly turned-over system

A photosynthate labelling method is presented which takes advantage of the natural difference in carbon-isotope composition () which exists between atmospheric CO₂ (-8) and commercially available compressed CO₂. Carbon dioxide with -4.0 and –27.9%., respectively, has been used for labelling. A plant growth cabinet served as the labelling compartment. CO₂-free air was continuously injected at a rate of up to 54m³·h^–1. Dilution of cabinet CO₂ by CO₂-free air was counterbalanced by addition of CO₂ with known constant . Since the labelling-cabinet atmosphere was continuously exchanged at a high rate, photosynthetic carbon-isotope discrimination was fully expressed. In order to study the distribution of carbon acquired by the plant during a defined growth period, the of CO₂ was modified by replacing, for example, atmospheric CO₂ by CO₂ with –27.9%. and the weight and 5 of plant carbon pools was monitored over time. In such an experiment the change of CO₂ was followed by a rapid change of the of sucrose in mature flag-leaf blades of wheat (Triticum aestivum L.). The 5 of sucrose stabilized near –51%., indicating complete exchange by current photosynthate. In contrast 83% of the total carbon in mature flag-leaf blades was not exchanged after 14 d continuous labelling. Differential labelling of pre- and post-anthesis photosynthate indicated that 13% of grain carbon originated from pre-anthesis photosynthesis. Carbon-isotope discrimination and its consideration in experimentation and labelling data evaluation are discussed in detail. Since the air supplied to the labelling cabinet is dry and free of CO₂, carbon-isotope discrimination and carbon turnover and partitioning can be studied over a wide range of CO₂ concentrations (0–2600 cm³ · m^–3) and vapor-pressure deficits.Abbreviation and Symbol PPFD photosynthetic photon flux density - carbon-isotope composition Dr. G. Schleser (Forschungszentrum Jülich, FRG) and Professor S. Hoernes (Mineralogisch-Petrologisches Institut, Universität Bonn) for valuable help and advice during the initial stages of the project and Professor W. Kühbauch (Institut für Pflanzenbau, Universität Bonn) for continuing support. Technical assistance of Ute Labusch, Petra Biermann, Ludwig Schmitz and Thomas Gebbing is gratefully acknowleged.

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Hierarchical clustering of 54 races and strains of the mulberry silkworm,Bombyx mori L: Significance of biochemical parameters

Summary A detailed analysis was undertaken to test the efficacy of hierarchical agglomerative clustering (UPGMA method) in grouping the races and strains of the mulberry silkworm, Bombyx moti L., and to ascertain the importance of biochemical parameters in the clustering process. The analysis was based on data from two rearing seasons with 54 selected races/strains of different geographic origin and varying yield potentials. The results indicate that seven clusters can be realised with yield parameters alone, whereas the inclusion of biochemical parameters in clustering resulted into two broad groups: one having all the breeds with high cocoon weight and shell weight, the other having all the low-yielding silkworm strains both from India and from other countries. Further sub-grouping under these two groups highlights genetical differences associated with the differentiation of various groups of races in temperate and tropical areas as well as their significance for silkworm breeding. Estimates of all ten variables were further subjected to quick clustering and the results showed that cluster 5, constituted by 38 lowyielding strains of India, China and Europe, had the highest values of the final cluster centre for amylase and the effective rate of rearing (ERR), while clusters 1 and 4 had the highest values for invertase and alkaline phosphatase. The evolutionary aspect of the genetic channelisation of silkworm races from various countries is discussed against the background of differences in the biochemical parameters and yield variables.     

Knockout of Nr2e3 prevents rod photoreceptor differentiation and leads to selective L-/M-cone photoreceptor degeneration in zebrafish

Mutations in the photoreceptor cell-specific nuclear receptor gene Nr2e3 increased the number of S-cone photoreceptors in human and murine retinas and led to retinal degeneration that involved photoreceptor and non-photoreceptor cells. The mechanisms underlying these complex phenotypes remain unclear. In the hope of understanding the precise role of Nr2e3 in photoreceptor cell fate determination and differentiation, we generated a line of Nr2e3 knockout zebrafish using CRISPR technology. In these Nr2e3-null animals, rod precursors undergo terminal mitoses but fail to differentiate as rods. Rod-specific genes are not expressed and the outer segment (OS) fails to form. Formation and differentiation of cone photoreceptors is normal. Specifically, there is no increase in the number of UV-cone or S-cone photoreceptors. Laminated retinal structure is maintained. After normal development, L-/M-cones selectively degenerate, with progressive shortening of OS that starts at age 1 month. The amount of cone phototransduction proteins is concomitantly reduced, whereas UV- and S-cones have normal OS lengths even at age 10 months. In vitro studies show Nr2e3 synergizes with Crx and Nrl to enhance rhodopsin gene expression. Nr2e3 does not affect cone opsin expression. Our results extend the knowledge of Nr2e3's roles and have specific implications for the interpretation of the phenotypes observed in human and murine retinas. Furthermore, our model may offer new opportunities in finding treatments for enhanced S-cone syndrome (ESCS) and other retinal degenerative diseases.     

The db mutation improves memory in younger mice in a model of Alzheimer's disease

Alzheimer's disease (AD) is the most common age-related neurodegenerative disease, while obesity is a major global public health problem associated with the metabolic disorder type 2 diabetes mellitus (T2DM). Chronic obesity and T2DM have been identified as invariant risk factors for dementia and late-onset AD, while their impacts on the occurrence and development of AD remain unclear. As shown in our previous study, the diabetic mutation (db, Lepr^db/db) induces mixed or vascular dementia in mature to middle-aged APP^ΔNL/ΔNL x PS1^P264L/P264L knock-in mice (db/AD). In the present study, the impacts of the db mutation on young AD mice at 10 weeks of age were evaluated. The db mutation not only conferred young AD mice with severe obesity, impaired glucose regulation and activated mammalian target of rapamycin (mTOR) signaling pathway in the mouse cortex, but lead to a surprising improvement in memory. At this young age, mice also had decreased cerebral Aβ content, which we have not observed at older ages. This was unlikely to be related to altered Aβ synthesis, as both β- and γ-secretase were unchanged. The db mutation also reduced the cortical IL-1β mRNA level and IBA1 protein level in young AD mice, with no significant effect on the activation of microglia and astrocytes. We conclude that the db mutation could transitorily improve the memory of young AD mice, a finding that may be partially explained by the relatively improved glucose homeostasis in the brains of db/AD mice compared to their counterpart AD mice, suggesting that glucose regulation could be a strategy for prevention and treatment of neurodegenerative diseases like AD.     

Novel MicroRNA-455-3p and its protective effects against abnormal APP processing and amyloid beta toxicity in Alzheimer's disease

The purpose of our study is to understand the protective role of miR-455-3p against abnormal amyloid precursor protein (APP) processing, amyloid beta (Aβ) formation, defective mitochondrial biogenesis/dynamics and synaptic damage in AD progression. In-silico analysis of miR-455-3p has identified the APP gene as a putative target. Using mutant APP cells, miR-455-3p construct, biochemical and molecular assays, immunofluorescence and transmission electron microscopy (TEM) analyses, we studied the protective effects of miR-455-3p on – 1) APP regulation, amyloid beta (Aβ)(1–40) & (1–42) levels, mitochondrial biogenesis & dynamics; 3) synaptic activities and 4) cell viability & apoptosis. Our luciferase reporter assay confirmed the binding of miR-455-3p at the 3’UTR of APP gene. Immunoblot, sandwich ELISA and immunostaining analyses revealed that the reduced levels of the mutant APP, Aβ(1–40) & Aβ(1–42), and C99 by miR-455-3p. We also found the reduced levels of mRNA and proteins of mitochondrial biogenesis (PGC1α, NRF1, NRF2, and TFAM) and synaptic genes (synaptophysin and PSD95) in mutant APP cells; on the other hand, mutant APP cells that express miR-455-3p showed increased mRNA and protein levels of biogenesis and synaptic genes. Additionally, expression of mitochondrial fission proteins (DRP1 and FIS1) were decreased while the fusion proteins (OPA1, Mfn1 and Mfn2) were increased by miR-455-3p. Our TEM analysis showed a decrease in mitochondria number and an increase in the size of mitochondrial length in mutant APP cells transfected with miR-455-3p. Based on these observations, we cautiously conclude that miR-455-3p regulate APP processing and protective against mutant APP-induced mitochondrial and synaptic abnormalities in AD.     

A novel indirubin derivative that increases somatic cell plasticity and inhibits tumorigenicity

Indirubin-based compounds affect diverse biological processes, such as inflammation and angiogenesis. In this study, we tested a novel indirubin derivative, LDD-1819 (2-((((2Z,3E)-5-hydroxy-5′-nitro-2′-oxo-[2,3′-biindolinylidene]-3-ylidene)amino)oxy)ethan-1-aminium chloride) for two major biological activities: cell plasticity and anti-cancer activity. Biological assays indicated that LDD-1819 induced somatic cell plasticity. LDD-1819 potentiated myoblast reprogramming into osteogenic cells and fibroblast reprogramming into adipogenic cells. Interestingly, in an assay of skeletal muscle dedifferentiation, LDD-1819 induced human muscle cellularization and blocked residual proliferative activity to produce a population of mononuclear refractory cells, which is also observed in the early stages of limb regeneration in urodele amphibians. In cancer cell lines, LDD-1819 treatment inhibited cell invasion and selectively induced apoptosis compared to normal cells. In an animal tumor xenograft model, LDD-1819 reduced human cancer cell metastasis in vivo at doses that did not produce toxicity. Biochemical assays showed that LDD-1819 possessed inhibitory activity against glycogen synthase kinase-3β, which is linked to cell plasticity, and aurora kinase, which regulates carcinogenesis. These results indicate that novel indirubin derivative LDD-1819 is a dual inhibitor of glycogen synthase kinase-3β and aurora A kinase, and has potential for development as an anti-cancer drug or as a reprogramming agent for cell-therapy based approaches to treat degenerative diseases.     

Influence of age on the bioaccumulation of heavy metals in Apodemus sylvaticus at Merja Zerga lagoon,Morocco

The influence of age and sex on the bioaccumulation of heavy metals in Apodemus sylvaticus was studied in Merja Zerga lagoon in northern Morocco. Five trace metal elements (Zn, Pb, Cr, Cu and Fe) were quantitatively analyzed by Varian AA 240 atomic absorption spectroscopy with graphite furnace in three organs (Liver, Kidney and Heart) from animals of different age and sex. The maximum metal level of the analyzed samples was recorded in adults and was limited to 46.62 μg/g for Pb and 35.1 μg/g for Cu, while it reached 22.69 μg/g, 7.59 μg/g and 6.78 μg/g for Cr, Zn and Fe, respectively. Highly significant differences were found for bioaccumulation of heavy metals according to animal ages and no significant differences were observed between the two sexes among the studied animals. Our results revealed also the existence of a strong correlation (r > 0.65) between the majority of biometric parameters and the trace element concentrations. In general, we found that age is a critical factor in estimating the level of heavy metal pollution. Other characteristics such as habitat, feeding habits and anti-predator behavior of the species need to be studied.     

Contributions of the C-terminal domain to poly(A)-specific ribonuclease (PARN) stability and self-association

Poly(A)-specific ribonuclease (PARN) catalyzes the degradation of mRNA poly(A) tail to regulate translation efficiency and mRNA decay in higher eukaryotic cells. The full-length PARN is a multi-domain protein containing the catalytic nuclease domain, the R3H domain, the RRM domain and the C-terminal intrinsically unstructured domain (CTD). The roles of the three well-structured RNA-binding domains have been extensively studied, while little is known about CTD. In this research, the impact of CTD on PARN stability and aggregatory potency was studied by comparing the thermal inactivation and denaturation behaviors of full-length PARN with two N-terminal fragments lacking CTD. Our results showed that K⁺ induced additional regular secondary structures and enhanced PARN stability against heat-induced inactivation, unfolding and aggregation. CTD prevented PARN from thermal inactivation but promoted thermal aggregation to initiate at a temperature much lower than that required for inactivation and unfolding. Blue-shift of Trp fluorescence during thermal transitions suggested that heat treatment induced rearrangements of domain organizations. CTD amplified the stabilizing effect of K⁺, implying the roles of CTD was mainly achieved by electrostatic interactions. These results suggested that CTD might dynamically interact with the main body of the molecule and release of CTD promoted self-association via electrostatic interactions.     

On the presence and localization of glycogen accumulations inside coronet cells of the saccus vasculosus of the dogfish (Scylliorhinus caniculus)

Summary In several coronet cells of the saccus vasculosus of Scylliorhinus large quantities of glycogen occur, as shown by light and electron microscopy. The significance of glycogen as an energy storage necessary for a transcellular ion transport process taking place in the coronet cells is discussed.The authors thank Dr. F.C.G. van de Veerdonk, W. F. Jansen and W. F. G. Flight for reading the manuscipt and for their critical remarks. They are also indebted to Mr. H. van Kooten and his staff for their valuable photographic assistance.