Identification and Functional Prediction of Large Intergenic Noncoding RNAs (lincRNAs) in Rainbow Trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

Long noncoding RNAs (lncRNAs) have been recognized in recent years as key regulators of diverse cellular processes. Genome-wide large-scale projects have uncovered thousands of lncRNAs in many model organisms. Large intergenic noncoding RNAs (lincRNAs) are lncRNAs that are transcribed from intergenic regions of genomes. To date, no lincRNAs in non-model teleost fish have been reported. In this report, we present the first reference catalog of 9674 rainbow trout lincRNAs based on analysis of RNA-Seq data from 15 tissues. Systematic analysis revealed that lincRNAs in rainbow trout share many characteristics with those in other mammalian species. They are shorter and lower in exon number and expression level compared with protein-coding genes. They show tissue-specific expression pattern and are typically co-expressed with their neighboring genes. Co-expression network analysis suggested that many lincRNAs are associated with immune response, muscle differentiation, and neural development. The study provides an opportunity for future experimental and computational studies to uncover the functions of lincRNAs in rainbow trout.     

THE STRUCTURAL ORGANIZATION OF THE SEPTATE AND GAP JUNCTIONS OF HYDRA

The septate junctions and gap junctions of Hydra were studied utilizing the extracellular tracers lanthanum hydroxide and ruthenium red. Analysis of the septate junction from four perspectives has shown that each septum consists of a single row of hexagons sharing common sides of 50–60 A. Each hexagon is folded into chair configuration. Two sets of projections emanate from the corners of the hexagons. One set (A projections) attaches the hexagons to the cell membranes at 80–100-A intervals, while the other set (V projections) joins some adjacent septa to each other. The septate junctions generally contain a few large interseptal spaces and a few septa which do not extend the full length of the junction. Basal to the septate junctions the cells in each layer are joined by numerous gap junctions. Gap junctions also join the muscular processes in each layer as well as those which connect the layers across the mesoglea. The gap junctions of Hydra are composed of rounded plaques 0.15–0.5 µ in diameter which contain 85-A hexagonally packed subunits. Each plaque is delimited from the surrounding intercellular space by a single 40-A band. Large numbers of these plaques are tightly packed, often lying about 20 A apart. This en plaque configuration of the gap junctions of Hydra contrasts with their sparser, more widely separated distribution in many vertebrate tissues. These studies conclude that the septate junction may possess some barrier properties and that both junctions are important in intercellular adhesion. On a morphological basis, the gap junction appears to be more suitable for intercellular coupling than the septate junction.     

Inhibition of leukotriene-induced contraction of guinea pig trachea by 5-lipoxygenase inhibitors

The effects of the 5-lipoxygenase inhibitors nordihydroguiaretic acid (NDGA), 5, 8, 11, 14-eicosatetraynoic acid (ETYA), 1-phenyl-3-pyrazolidone (phenidone) and BW-755c, on the contractile response to LTC4 or LTD4 were examined on the isolated guinea pig trachea. Responses to either LTC4 or LTD4 were obtained on indomethacin treated tissues, in the presence of either L-serine-borate complex or L-cysteine, respectively, to inhibit metabolic conversion of the leukotrienes. NDGA (30 microM) and ETYA (100 microM) produced a selective competitive antagonism of LTD4-induced contractions, while phenidone antagonized both LTC4- and LTD4-induced responses in a non-competitive manner. In contrast, BW-755c (30 microM) did not significantly antagonize LTC4 or LTD4 concentration-response curves. The results suggest that leukotriene antagonism may be produced by large concentrations of some 5-lipoxygenase inhibitors.     

Arrestment of carbohydrate metabolism during anaerobic dormancy and aerobic acidosis inArtemia embryos: determination of pH-sensitive control points

Summary Changes in concentrations of trehalose, glycogen, glycerol, some glycolytic intermediates and adenylate nucleotides that occur during aerobic development have been compared to those seen during anaerobic dormancy and aerobic acidosis in gastrula-stage embryos ofArtemia. The latter two incubation conditions are known to foster large drops in intracellular pH (Busa et al. 1982; Busa and Crowe 1983).During aerobic development, trehalose levels decline while glycogen and glycerol are synthesized (Fig. 1). These transitions are blocked during both anaerobic dormancy and aerobic acidosis, but are resumed by return of embryos to aerobic incubation (Fig. 1). Thus, it is concluded that carbohydrate catabolism in hydrated embryos is directly modulated by intracellular pH. Changes in metabolite levels (Figs. 2–4) reveal that this process is controlled primarily at the trehalase and hexokinase reactions with a less-pronounced negative crossover point noted at the phosphofructokinase step. Each of these reactions is shown to be nonequilibrium by comparing the mass action ratio to the equilibrium constant (Table 1).When embryos are placed under anaerobic conditions, ATP levels drop dramatically while AMP increases in concentration (Fig. 5). These changes are reflected in a drop in adenylate energy charge from a control value of 0.73 to 0.42 (Fig. 6). Aerobic acidosis only leads to a slight decrease in energy charge, emphasizing that shifts in adenylate poolsAbbreviations pH _i intracellular pH - glyceraldehyde 3-P glyeraldehyde 3-phosphate - DHAP dihydroxyacetone phosphate - glucose 6-P glucose 6-phosphate - fructose 6-P fructose 6-phosphate - fructose 1,6-P ₂ fructose 1,6-bisphosphate - HK hexokinase - PFK phosphofructokinase - MAR mass action ratio - K _eq equilibrium constant     

Activity and distribution of lysosomal enzymes during collagenous matrix-induced cartilage,bone, and bone marrow development

The appearance of the lysosomal enzymes acid phosphatase, arylsulfatase, and β-glucuronidase was studied during endochondral bone and bone marrow formation induced by implantation of demineralized bone matrix. The activities of acid phosphatase and β-glucuronidase gradually increased from the stage of mesenchymal cell proliferation on Day 3 onward to reach a peak on Day 13, during maximal bone remodeling. However, arysulfatase activity exhibited a sharp increase on Day 9, associated with the onset of cartilage hypertrophy and chondrolysis. The peak of arylsulfatase activity was also attained on Day 13. The activities of all three enzymes declined on Day 15 but acid phosphatase again exhibited an increase during hematopoietic bone marrow differentiation on Days 19–21. Histochemical and ultrastructural studies revealed intense lysosomal enzyme activity in macrophage-like cells on Day 7 and thereafter. During chondrolysis and bone remodeling, these cells were present in a perivascular location. Osteoclasts also exhibited strong reactivity for the lysosomal enzymes. Due to its characteristic temporal appearance during development of endochondral bone, arysulfatase may be used as a marker enzyme for chondrolysis and bone resorption.     

Overexpression of Acetohydroxyacid Synthase from Arabidopsis as an Inducible Fusion Protein in Escherichia coli: Production of Polyclonal Antibodies, and Immunological Characterization of the Enzyme

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) is the first enzyme unique to the biosynthesis of valine, leucine, and isoleucine. This enzyme is the target site of several classes of structurally unrelated herbicides. The conventional method of antibody production using purified protein has not been successful with this enzyme. Two separate fragments of a gene encoding a portion of the mature region of AHAS from Arabidopsis were fused with the trpE gene from Escherichia coli using the pATH1 vector. E. coli cells transformed with each respective plasmid expressed a fusion protein at levels greater than 10% of the total cell protein. The fusion protein was purified and used to immunize rabbits. Antisera obtained from the immunized rabbits immunoprecipitated AHAS activity from Arabidopsis cell free extracts. The anti-AHAS antisera reacted with a 65 kilodalton protein band in electrophoretically resolved extracts of Arabidopsis. In cross-reactivity tests, this antibody was able to immunoprecipitate AHAS activity from various plant species. Furthermore, a protein band with a molecular mass of 65 kilodaltons was detected in the crude extracts of all plant species tested on a Western blot. These results indicate that the 65 kilodalton protein represents AHAS in various plant species. The wide spectrum of cross-reactivity for the antisera supports the view that the AHAS enzyme is highly conserved across all plant species.     

Localization and enumeration of fimbria-associated adhesins of Bacteroides loescheii.

Monoclonal antibodies that specifically inhibit coaggregation between Bacteroides loescheii PK1295 and its two gram-positive partners Streptococcus sanguis 34 and Actinomyces israelii PK14 were used to enumerate and localize two distinct types of fimbria-associated adhesins on the surface of B. loescheii. Binding studies with radiolabeled monoclonal antibodies indicated that a maximum (Bmax calculated from Scatchard plots) of approximately 400 adhesin molecules specific for S. sanguis and 310 adhesin molecules specific for A. israelii reside on the surface of the cell. Immunoelectron microscopy revealed that the adhesins were not an integral part of the fimbrial subunit; rather, they were usually found on the distal portion of the structures arranged in a random fashion.     

Ketamine Does Not Produce Relief of Neuropathic Pain in Mice Lacking the β-Common Receptor (CD131)

Neuropathic pain (NP) is a debilitating condition associated with traumatic, metabolic, autoimmune and neurological etiologies. Although the triggers for NP are diverse, there are common underlying pathways, including activation of immune cells in the spinal cord and up-regulation of the N-methyl-D-aspartate receptor (NMDAR). Ketamine, a well-known NDMAR antagonist, reduces neuropathic pain in a sustained manner. Recent study has shown that the novel 11-amino acid peptide erythropoietin derivative ARA290 produces a similar, long-lasting relief of NP. Here, we show that both drugs also have similar effects on the expression of mRNA of the NMDAR, as well as that of microglia, astrocytes and chemokine (C-C motif) ligand 2, all-important contributors to the development of NP. Although the effects of ketamine and ARA 290 on NP and its molecular mediators suggest a common mechanism of action, ARA 290 has no affinity for the NMDAR and acts specifically via the innate repair receptor (IRR) involved in tissue protection. We speculated therefore, that the IRR might be critically involved in the action of ketamine on neuropathic pain. To evaluate this, we studied the effects of ketamine and ARA 290 on acute pain, side effects, and allodynia following a spared nerve injury model in mice lacking the β-common receptor (βcR), a structural component of the IRR. Ketamine (50 mg/kg) and ARA 290 (30 µg/kg) produced divergent effects on acute pain: ketamine produced profound antinociception accompanied with psychomotor side effects, but ARA290 did not, in both normal and knock out mice. In contrast, while both drugs were antiallodynic in WT mice, they had no effect on NP in mice lacking the βcR. Together, these results show that an intact IRR is required for the effective treatment of NP with either ketamine or ARA 290, but is not involved in ketamine’s analgesic and side effects.