Sequential colonization by river periphyton analysed by microscopy and molecular fingerprinting

1. An artificial glass substratum was incubated in the River Danube for a period of 28 days in order to detect the sequential colonization of microorganisms.
2. Light and fluorescent microscopy showed that microalgae and the picoalgal fraction on the slides increased rapidly over the first 2 weeks of colonization. Diatoms were numerically the most abundant component of the periphyton and their species richness and diversity increased rapidly in the early phase of colonization whereas diversity subsequently increased moderately.
3. Evenness of the diatom community was initially high, lower in the intermediate phase and again higher later on. Succession involving early, intermediate and late colonizer species was observed. Community composition during the first 5 days of colonization was very different from later stages whereas there were only minor changes subsequently.
4. Molecular community analysis by means of terminal restriction fragment length polymorphism analysis of PCR amplified 16S rRNA and 18S rRNA genes pointed to even larger differences between the composition of samples obtained early and late in the period.
5. The number of 18S rRNA and 16S rRNA terminal restriction fragments (T-RF-s) was variable over the colonization period and the fragment patterns of both the bacterial and eukaryotic portion of the microbial community were variable, with most T-RF-s unique to a single sample, suggesting a wide diversity and dynamic properties of periphytic organisms.     

Molecular,cytological and morpho-agronomical characterization of hexaploid somatic hybrids in Medicago

Somatic hybrid plants produced by protoplast fusion between tetraploid Medicago sativa (2n= 4x=32) and the diploid species Medicago coerulea (2n= 2x=16) have been RFLP fingerprinted to establish their nuclear composition. Although all of the chromosomes were present, molecular analysis revealed an incomplete incorporation of the alleles of the diploid parent in the fusion products. In the polycross progeny the alleles of both parents segregated in a Mendelian mode. Cytological observations indicated that in the somatic hybrid population minor abnormalities are present; these are restricted mainly to the formation of univalents and lagging chromosomes. Meiosis appeared to be more stable than has been previously reported in the hexaploids of alfalfa. The somatic hybrids grown in the field had a rather vigorous aspect, particularly with respect to the vegetative organs. Forage yield was comparable to that of thmore productive parent. The results are discussed with a view to utilizing the somatic hybrids as starting material for breeding alfalfa at the hexaploid level.This paper was supported by the National Research Council of Italy, Special Project RAISA, Sub-project No.2 paper No. 1911     

Concerted transpositions of mobile genetic elements coupled with fitness changes in Drosophila melanogaster

In an inbred low-activity (LA) strain of Drosophila melanogaster with a low level of fitness and a complex of inadaptive characters, in situ hybridization reveals an invariant pattern of distribution of three copia-like elements (mdg-1, mdg-3, and copia). Rare, spontaneous, multiple transpositions of mobile elements in the LA strain were shown to be coupled with a drastic increase of fitness. A changed pattern of various types of mobile elements was also observed on selecting the LA strain for higher fitness. High-fitness strains show transpositions of mobile elements to definite chromosomal sites ("hot spots"). Concerted changes in the location of three different mobile elements were found to be coupled with an increase of fitness. The mdg-1 distribution patterns were also examined in two low-fitness strains independently selected from the high-fitness ones. Fitness decrease was accompanied by mdg-1 excision from the hot spots of their location usually detected in the high-fitness strains. The results suggest the existence of a system of adaptive transpositions of mobile elements that takes part in fitness control.      

Oxidized Phospholipid OxPAPC Activates TRPA1 and Contributes to Chronic Inflammatory Pain in Mice

Oxidation products of the naturally occurring phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphatidylcholine (PAPC), which are known as OxPAPC, accumulate in atherosclerotic lesions and at other sites of inflammation in conditions such as septic inflammation and acute lung injury to exert pro- or anti-inflammatory effects. It is currently unknown whether OxPAPC also contributes to inflammatory pain and peripheral neuronal excitability in these conditions. Here, we observed that OxPAPC dose-dependently and selectively activated human TRPA1 nociceptive ion channels expressed in HEK293 cells in vitro, without any effect on other TRP channels, including TRPV1, TRPV4 and TRPM8. OxPAPC agonist activity was dependent on essential cysteine and lysine residues within the N-terminus of the TRPA1 channel protein. OxPAPC activated calcium influx into a subset of mouse sensory neurons which were also sensitive to the TRPA1 agonist mustard oil. Neuronal OxPAPC responses were largely abolished in neurons isolated from TRPA1-deficient mice. Intraplantar injection of OxPAPC into the mouse hind paw induced acute pain and persistent mechanical hyperalgesia and this effect was attenuated by the TRPA1 inhibitor, HC-030031. More importantly, we found levels of OxPAPC to be significantly increased in inflamed tissue in a mouse model of chronic inflammatory pain, identified by the binding of an OxPAPC-specific antibody. These findings suggest that TRPA1 is a molecular target for OxPAPC and OxPAPC may contribute to chronic inflammatory pain through TRPA1 activation. Targeting against OxPAPC and TRPA1 signaling pathway may be promising in inflammatory pain treatment.     

Ongoing speciation within the Anastrepha fraterculus cryptic species complex: the case of the Andean morphotype

The Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) cryptic species complex is currently composed of seven taxonomically recognized morphotypes. Both, pre‐ and post‐zygotic isolation has been documented among four of these morphotypes, revealing that in fact they appear to be distinct biological entities. In order to progress in the full delimitation of species within the complex, we examined reproductive isolation between a Colombian population of the Andean morphotype and populations belonging to four other morphotypes spanning from Mexico to Argentina. Flies from the Andean morphotype exhibited strong pre‐zygotic mating isolation through temporal partitioning of mating activity. Post‐zygotic isolation was observed for crosses of males of all morphotypes and Andean morphotype females, yet most of the F1 hybrid ♂ × F1 hybrid ♀ self‐crosses showed normal levels of fertility, a finding suggesting a nuclear–cytoplasmic interaction according to previous studies. Overall, the Andean morphotype within the complex also appears to be a distinct biological entity. We discuss the implications of these findings for the understanding of speciation mechanisms in the Neotropical genus Anastrepha.     

Vesicle-associated Membrane Protein 2 (VAMP2) but Not VAMP3 Mediates cAMP-stimulated Trafficking of the Renal Na+-K+-2Cl? Co-transporter NKCC2 in Thick Ascending Limbs

In the kidney, epithelial cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na⁺/K⁺/2Cl⁻ co-transporter NKCC2. Steady-state surface NKCC2 levels in the apical membrane are maintained by a balance between exocytic delivery, endocytosis, and recycling. cAMP is the second messenger of hormones that enhance NaCl absorption. cAMP stimulates NKCC2 exocytic delivery via protein kinase A (PKA), increasing steady-state surface NKCC2. However, the molecular mechanism involved has not been studied. We found that several members of the SNARE family of membrane fusion proteins are expressed in TALs. Here we report that NKCC2 co-immunoprecipitates with VAMP2 in rat TALs, and they co-localize in discrete domains at the apical surface. cAMP stimulation enhanced VAMP2 exocytic delivery to the plasma membrane of renal cells, and stimulation of PKA enhanced VAMP2-NKCC2 co-immunoprecipitation in TALs. In vivo silencing of VAMP2 but not VAMP3 in TALs blunted cAMP-stimulated steady-state surface NKCC2 expression and completely blocked cAMP-stimulated NKCC2 exocytic delivery. VAMP2 was not involved in constitutive NKCC2 delivery. We concluded that VAMP2 but not VAMP3 selectively mediates cAMP-stimulated NKCC2 exocytic delivery and surface expression in TALs. We also demonstrated that cAMP stimulation enhances VAMP2 exocytosis and promotes VAMP2 interaction with NKCC2.     

LIM kinase 1 and cofilin regulate actin filament population required for dynamin-dependent apical carrier fission from the trans-Golgi network

The functions of the actin cytoskeleton in post-Golgi trafficking are still poorly understood. Here, we report the role of LIM Kinase 1 (LIMK1) and its substrate cofilin in the trafficking of apical and basolateral proteins in Madin-Darby canine kidney cells. Our data indicate that LIMK1 and cofilin organize a specialized population of actin filaments at the Golgi complex that is selectively required for the emergence of an apical cargo route to the plasma membrane (PM). Quantitative pulse-chase live imaging experiments showed that overexpression of kinase-dead LIMK1 (LIMK1-KD), or of LIMK1 small interfering RNA, or of an activated cofilin mutant (cofilin S3A), selectively slowed down the exit from the trans-Golgi network (TGN) of the apical PM marker p75-green fluorescent protein (GFP) but did not interfere with the apical PM marker glycosyl phosphatidylinositol-YFP or the basolateral PM marker neural cell adhesion molecule-GFP. High-resolution live imaging experiments of carrier formation and release by the TGN and analysis of peri-Golgi actin dynamics using photoactivatable GFP suggest a scenario in which TGN-localized LIMK1-cofilin regulate a population of actin filaments required for dynamin-syndapin-cortactin–dependent generation and/or fission of precursors to p75 transporters.