mTOR kinase is needed for the development and stabilization of dendritic arbors in newly born olfactory bulb neurons

Neurogenesis is the process of neuron generation, which occurs not only during embryonic development but also in restricted niches postnatally. One such region is called the subventricular zone (SVZ), which gives rise to new neurons in the olfactory bulb (OB). Neurons that are born postnatally migrate through more complex territories and integrate into fully functional circuits. Therefore, differences in the differentiation of embryonic and postnatally born neurons may exist. Dendritogenesis is an important process for the proper formation of future neuronal circuits. Dendritogenesis in embryonic neurons cultured in vitro was shown to depend on the mammalian target of rapamycin (mTOR). Still unknown, however, is whether mTOR could regulate the dendritic arbor morphology of SVZ‐derived postnatal OB neurons under physiological conditions in vivo. The present study used in vitro cultured and differentiated SVZ‐derived neural progenitors and found that both mTOR complex 1 and mTOR complex 2 were required for the dendritogenesis of SVZ‐derived neurons. Furthermore, using a combination of in vivo electroporation of neural stem cells in the SVZ and genetic and pharmacological inhibition of mTOR, it was found that mTOR was crucial for the growth of basal and apical dendrites in postnatally born OB neurons under physiological conditions and contributed to the stabilization of their basal dendrites. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1308–1327, 2016     

Subsarcolemmal mitochondria isolated with the proteolytic enzyme nagarse exhibit greater protein specific activities and functional coupling

Skeletal muscle mitochondria are arranged as a reticulum. Insight into the functional characteristics of such structure is achieved by viewing the network as consisting of “subsarcolemmal” (SS) and “intermyofibrillar” (IMF) regions. During the decades, most, but not all, published studies have reported higher (sometimes over 2-fold) enzyme and enzyme-pathway protein-specific activities in IMF compared to SS mitochondria. We tested the hypothesis that non-mitochondrial protein contamination might account for much of the apparently lower specific activities of isolated SS mitochondria. Mouse gastrocnemii (n=6) were suspended in isolation medium, minced, and homogenized according to procedures typically used to isolate SS mitochondria. However, the supernatant fraction, collected after the first slow-speed (800g) centrifugation, was divided equally: one sample was exposed to nagarse (MITO+), while the other was not (MITO?). Nagarse treatment reduced total protein yield by 25%, while it increased protein-specific respiration rates (nmol O₂ min^?1 mg^?1), by 38% under “resting” (state 4) and by 84% under maximal (state 3) conditions. Nagarse therefore increased the respiratory control ratio (state 3/state 4) by 30%. In addition, the ADP/O ratio was increased by 9% and the activity of citrate synthase (U/mg) was 49% higher. Mass spectrometry analysis indicated that the MITO+ preparation contained less contamination from non-mitochondrial proteins. We conclude that nagarse treatment of SS mitochondria removes not only non-mitochondrial proteins but also the protein of damaged mitochondria, improves indices of functional integrity, and the resulting protein-specific activities.     

Analysis of low density lipoprotein receptor gene mutations and microsatellite haplotypes in Greek FH heterozygous children: six independent ancestors account for 60% of probands

This study reports the characterization of 60% of low density lipoprotein receptor (LDLR) gene mutations in 150unrelated Greek familial hypercholes-terolaemia (FH) heterozygous children by the analysis of six LDLR gene mutations. The linkage disequilibrium of two polymorphic microsatellites (D19S394 and D19S221) flanking the LDLR gene on chromosome19 to the four most common mutations strongly suggests that each mutation is identical-by-descent in the probands included in this study (this is also supported by the geographical distribution of FH families with these mutations throughout Greece) and permits an estimation of the number of generations from a common ancestor for each mutation. The characterization of 60% of LDLR mutations in a representative sample of Greek FH heterozygotes provides a basis for the diagnosis of FH through DNA analysis in Greece, by using single-strand conformation polymorphism analysis followed by allele-specific oligonucleotide hybridization (exon6 mutations) or restriction endonuclease analysis (C152R, V408M). A rapid diagnostic assay positive for the mutation has been developed for the most common mutation, G528D. The application of simple DNA diagnostic assays for LDLR mutation analysis are appropriate for the early identification of FH heterozygotes in Greece and are useful for the primary prevention of coronary artery disease. Received: 7 July 1997 / Accepted: 5 November 1997     

An Apparatus (Georgiou-Petri Dish) for Growing Fungi and other Microorganisms on Liquid Media in a Petri Dish

This work describes a new apparatus for growing fungi and other microorganisms on liquid nutrient media in a Petri dish. The apparatus is composed of a net supporting a cellophane membrane stretched between an outer and an inner ring that is placed inside a Petri dish. This modification of the standard Petri dish offers many advantages for studying growth, metabolism, differentiation, and other aspects of fungi in liquid cultures with minimal waste of expensive chemicals. Monitoring of excreted or absorbed substances by the fungi, the aseptic transfer of undisturbed fungal colonies from dish to dish, and harvesting are made easier, using this apparatus.     

Apical cells of brown algae with particular reference to Sphacelariales, Dictyotales and Fucales

Brown algae show a significant diversity in thallus forms, giving a great number of model systems for the study of many important morphogenetic mechanisms. Thallus growth in brown algae is diffuse, intercalary or apical. The latter takes place by means of one or more apical cells. Among the brown algal groups, Sphacelariales, Dictyotales and Fucales give the best examples of apical growth, and have been repeatedly used for the study of the morphogenetic role of apical cells. In Sphacelariales the apical cells appear strongly polarized, the polarity expressed also on the organization of the microtubule cytoskeleton. These cells show a type of growth that can be compared with tip growth of root hairs, moss protonemata, pollen tubes and fungal hyphae, and is called ‘tip-like growth’. The thallus of Dictyotales grows by the activity of one or more apical cells showing variable degree of polarity. These cells do not exhibit any type of apical growth. In Fucales the vegetative thallus develops by means of an active apical meristem, which includes a large apical cell. This cell does not show polar organization or apical growth. However, in germinating zygotes of Fucales a polar axis is established and during the first stages of development they show a typical tip growth. In the present paper, the available information on the structure and division pattern of apical cells is presented. Their morphogenetic role is discussed, in relation to polarity, cytoskeleton organization, and apical dominance.     

Pseudomonas cepacia lipase: Esterification reactions in AOT microemulsion systems

Summary The activity of purifiedPseudomonas cepacia lipase has been investigated in esterification reactions of various aliphatic alcohols with natural fatty acids. The reactions were carried out in microemulsions formed in isooctane by bis(2ethylhexyl)sulfosuccinate sodium salt (AOT). The optima pH, T and water content (w_o) for the enzyme activity in this type of microemulsions have been determined. Studies on the effect of various fatty acids and alcohols on the enzyme specificity have shown a preference of this lipase for palmitic and caprylic acid as well as for propanol, while reactions involving cyclic alcohols can not be catalyzed at all. The differences on the behavior of this lipase as compared to other lipases studied in microemulsion systems as well as in other systems are discussed.     

Induction of somatic and male crossing-over by bleomycin in Drosophila melanogaster

Bleomycin (BLM) is well known as an antibiotic as well as for its antineoplastic activity. A clinical preparation of BLM was tested for its recombinogenicity in a higher eukaryotic organism, Drosophila melanogaster. Feeding of the F1 larvae on a medium with BLM increased somatic crossing-over spots on female tergites and induced recombination in male germ cells. However, nonlinear dose-response curves were obtained. Malformed tergites were also observed in females treated with BLM.