Versatility of microglial bioenergetic machinery under starving conditions

Microglia are highly dynamic cells in the brain. Their functional diversity and phenotypic versatility brought microglial energy metabolism into the focus of research. Although it is known that microenvironmental cues shape microglial phenotype, their bioenergetic response to local nutrient availability remains unclear.In the present study effects of energy substrates on the oxidative and glycolytic metabolism of primary – and BV-2 microglial cells were investigated. Cellular oxygen consumption, glycolytic activity, the levels of intracellular ATP/ADP, autophagy, mTOR phosphorylation, apoptosis and cell viability were measured in the absence of nutrients or in the presence of physiological energy substrates: glutamine, glucose, lactate, pyruvate or ketone bodies.All of the oxidative energy metabolites increased the rate of basal and maximal respiration. However, the addition of glucose decreased microglial oxidative metabolism and glycolytic activity was enhanced. Increased ATP/ADP ratio and cell viability, activation of the mTOR and reduction of autophagic activity were observed in glutamine-supplemented media. Moreover, moderate and transient oxidation of ketone bodies was highly enhanced by glutamine, suggesting that anaplerosis of the TCA-cycle could stimulate ketone body oxidation.It is concluded that microglia show high metabolic plasticity and utilize a wide range of substrates. Among them glutamine is the most efficient metabolite. To our knowledge these data provide the first account of microglial direct metabolic response to nutrients under short-term starvation and demonstrate that microglia exhibit versatile metabolic machinery. Our finding that microglia have a distinct bioenergetic profile provides a critical foundation for specifying microglial contributions to brain energy metabolism.     

Identification and re-addressing of a transcriptionally permissive locus in the porcine genome

Recently, we established the Sleeping Beauty transposon system for germ line competent transgenesis in the pig. Here, we extend this approach to re-target a transposon-tagged locus for a site-specific gene knock-in, and generated a syngeneic cohort of piglets carrying either the original transposon or the re-targeted event. A Cre-loxP-mediated cassette exchange of the tagging transposon with a different reporter gene was performed, followed by flow cytometric sorting and somatic cell nuclear transfer of recombined cells. In parallel, the original cells were employed in somatic cell nuclear transfer to generate clone siblings, thereby resulting in a clone cohort of piglets carrying different reporter transposons at an identical chromosomal location. Importantly, this strategy supersedes the need for an antibiotic selection marker. This approach expands the arsenal of genome engineering technologies in domestic animals, and will facilitate the development of large animal models for human diseases. Potentially, the syngeneic cohort of pigs will be instrumental for vital tracking of transplanted cells in pre-clinical assessments of novel cell therapies.     

A survey of scale insects in soil samples from Europe (Hemiptera,Coccomorpha)

In the last decades, several expeditions were organized in Europe by the researchers of the Hungarian Natural History Museum to collect snails, aquatic insects and soil animals (mites, springtails, nematodes, and earthworms). In this study, scale insect (Hemiptera: Coccomorpha) specimens extracted from Hungarian Natural History Museum soil samples (2970 samples in total), all of which were collected using soil and litter sampling devices, and extracted by Berlese funnel, were examined. From these samples, 43 scale insect species (Acanthococcidae 4, Coccidae 2, Micrococcidae 1, Ortheziidae 7, Pseudococcidae 21, Putoidae 1 and Rhizoecidae 7) were found in 16 European countries. In addition, a new species belonging to the family Pseudococcidae, Brevennia larvalis Kaydan, sp. n. and a new species of Ortheziidae, Ortheziola editae Szita & Konczné Benedicty, sp. n. are described and illustrated based on the adult female stage. Revised keys to the adult females of Brevennia and Ortheziola are presented.     

Purification, crystallization and X-ray diffraction analyses of the T. elongatus PSII core dimer with strontium replacing calcium in the oxygen-evolving complex

The core complex of photosystem II (PSII) was purified from thermophilic cyanobacterium Thermosynechococcus elongatus grown in Sr(2+)-containing and Ca(2+)-free medium. Functional in vivo incorporation of Sr(2+) into the oxygen-evolving complex (OEC) was confirmed by EPR analysis of the isolated and highly purified SrPSII complex in agreement with the previous study of Boussac et al. [J. Biol. Chem. 279 (2004) 22809-22819]. Three-dimensional crystals of SrPSII complex were obtained which diffracted to 3.9 A and belonged to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a=133.6 A, b=236.6 A, c=307.8 A. Anomalous diffraction data collected at the Sr K-X-ray absorption edge identified a novel Sr(2+)-binding site which, within the resolution of these data (6.5 A), is consistent with the positioning of Ca(2+) in the recent crystallographic models of PSII [Ferreira et al. Science 303 (2004) 1831-1838, Loll et al. Nature 438 (2005) 1040-1044]. Fluorescence measurements on SrPSII crystals confirmed that crystallized SrPSII was active in transferring electrons from the OEC to the acceptor site of the reaction centre. However, SrPSII showed altered functional properties of its modified OEC in comparison with that of the CaPSII counterpart: slowdown of the Q(A)-to-Q(B) electron transfer and stabilized S(2)Q(A)(-) charge recombination.     

The sensitivity of Photosystem II to damage by UV-B radiation depends on the oxidation state of the water-splitting complex

The water-oxidizing complex of Photosystem II is an important target of ultraviolet-B (280-320 nm) radiation, but the mechanistic background of the UV-B induced damage is not well understood. Here we studied the UV-B sensitivity of Photosystem II in different oxidation states, called S-states of the water-oxidizing complex. Photosystem II centers of isolated spinach thylakoids were synchronized to different distributions of the S(0), S(1), S(2) and S(3) states by using packages of visible light flashes and were exposed to UV-B flashes from an excimer laser (lambda=308 nm). The loss of oxygen evolving activity showed that the extent of UV-B damage is S-state-dependent. Analysis of the data obtained from different synchronizing flash protocols indicated that the UV-sensitivity of Photosystem II is significantly higher in the S(3) and S(2) states than in the S(1) and S(0) states. The data are discussed in terms of a model where UV-B-induced inhibition of water oxidation is caused either by direct absorption within the catalytic manganese cluster or by damaging intermediates of the water oxidation process.     

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     

1-[4-(2-Aminoethoxy)phenylcarbonyl]-3,5-bis-(benzylidene)-4-oxopiperidines: a novel series of highly potent revertants of P-glycoprotein associated multidrug resistance

The 1-[4-(2-aminoethoxy)phenylcarbonyl]-3,5-bis-(benzylidene)-4-oxopiperidines 5-8 are a novel cluster of highly potent P-glycoprotein dependent multidrug resistance (MDR) revertants. Using a concentration of 4mug/mL, these compounds possess 11-43 times the potency of verapamil in reversing MDR in murine L-5178 lymphoma cells transfected with the human MDR1 gene. Structure-activity relationships reveal that the attachment of the N-aroyl group to various 3,5-bis(benzylidene)-4-piperidones is essential for MDR reversal to occur. In terms of potencies, the 1-piperidinyl group is the preferred terminal amine while the 4-methyl and 4-chloro substituents are the optimal groups for placement in the benzylidene aryl rings.     

Methylation of chloroplast DNA does not affect viability and maternal inheritance in tobacco and may provide a strategy towards transgene containment

We report the integration of a type II restriction-methylase, mFokI, into the tobacco chloroplast genome and we demonstrate that the introduced enzyme effectively directs the methylation of its target sequence in vivo and does not affect maternal inheritance. We further report the transformation of tobacco with an E. coli dcm methylase targeted to plastids and we demonstrate efficient cytosine methylation of the plastid genome. Both adenosine methylation of FokI sites and cytosine methylation of dcm sites appeared phenotypically neutral. The ability to tolerate such plastid genome methylation is a pre-requisite for a proposed plant transgene containment system. In such a system, a chloroplast located, maternally inherited restriction methylase would provide protection from a nuclear-encoded, plastid targeted restriction endonuclease. As plastids are not paternally inherited in most crop species, pollen from such plants would carry the endonuclease transgene but not the corresponding methylase; the consequence of this should be containment of all nuclear transgenes, as pollination will only be viable in crosses to the appropriate transplastomic maternal background.