Effects of 5-hydroxytryptamine on the neuronal firing rate of bulbar reticular neurons

The effects of 5-hydroxytryptamine (5-HT) on neuronal firing rate were studied in the reticular gigantocellular nucleus (GRN) and, for a comparison, in the interstitial (IRN), the parvicellular (PRN) and the lateral (LRN) nuclei, sharing some of GRN functional characteristics. Unitary extracellular recordings performed in anesthetized rats demonstrated that microiontophoretic application of 5-HT modulated the background firing rate in 92% of GRN, in 100% of IRN and LRN, and in 77% of PRN tested neurons. In GRN, 5-HT application induced excitatory responses in 49% of the neurons tested and inhibitions in 43% of them. Both types of effects were dose dependent and appeared scattered throughout the nucleus. Enhancements and decreases of firing rate in response to 5-HT application were also recorded in IRN (58% and 42% respectively), LRN (43% and 57%) and PRN (36% and 41%). The 5-HT(1A) receptor agonist 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT) mimicked 5-HT evoked inhibitions in all the nuclei tested and induced weak inhibitory responses also in neurons excited by 5-HT. The 5-HT2A receptor agonist alphamethyl-5-hydroxytryptamine (alpha-me-5-HT) mimicked excitatory as well as inhibitory responses to 5-HT, the former prevailing in GRN and the latter in the remaining reticular nuclei. Both excitatory and inhibitory responses to 5-HT were partially or totally blocked by the application of 5-HT2 receptor antagonist ketanserin. It is concluded that an extended, strong and differentiated control is exerted by 5-HT on the electrical activity of bulbar reticular neurons. Both 5-HT(1A) and 5-HT(2A) receptors mediate these effects, but the involvement of other receptors appears probable.     

Peptide-linked molecular beacons for efficient delivery and rapid mRNA detection in living cells

Real-time visualization of specific endogenous mRNA expression in vivo has the potential to revolutionize medical diagnosis, drug discovery, developmental and molecular biology. However, conventional liposome- or dendrimer-based cellular delivery of molecular probes is inefficient, slow, and often detrimental to the probes. Here we demonstrate the rapid and sensitive detection of RNA in living cells using peptide-linked molecular beacons that possess self-delivery, targeting and reporting functions. We conjugated the TAT peptide to molecular beacons using three different linkages and demonstrated that, at relatively low concentrations, these molecular beacon constructs were internalized into living cells within 30 min with nearly 100% efficiency. Further, peptide-based delivery did not interfere with either specific targeting by or hybridization-induced fluorescence of the probes. We could therefore detect human GAPDH and survivin mRNAs in living cells fluorescently, revealing intriguing intracellular localization patterns of mRNA. We clearly demonstrated that cellular delivery of molecular beacons using the peptide-based approach has far better performance compared with conventional transfection methods. The peptide-linked molecular beacons approach promises to open new and exciting opportunities in sensitive gene detection and quantification in vivo.     

Quantification of the virus-host interaction in human T lymphotropic virus I infection

Background

Cellular infection with human immunodeficiency virus (HIV) both in vitro and in vivo requires a member of the chemokine receptor family to act as a co-receptor for viral entry. However, it is presently unclear to what extent the interaction of HIV proteins with chemokine receptors generates intracellular signals that are important for productive infection.

Results

In this study we have used a recently described family of chemokine inhibitors, termed BSCIs, which specifically block chemokine-induced chemotaxis without affecting chemokine ligands binding to their receptors. The BSCI termed Peptide 3 strongly inhibited CCR5 mediated HIV infection of THP-1 cells (83 ± 7% inhibition assayed by immunofluoresence staining), but had no effect on gp120 binding to CCR5. Peptide 3 did not affect CXCR4-dependent infection of Jurkat T cells.

Conclusion

These observations suggest that, in some cases, intracellular signals generated by the chemokine coreceptor may be required for a productive HIV infection.     

Estrogen receptors in rabbit thymus

The estrogen receptors presence or absence in the thymus gland represents the basic element to assert or to exclude any estrogenic action in the thymus. Now we have measured the presence of cytoplasmatic estrogen receptors in the female rabbits thymus, using some methods (sucrose gradient, protamine sulphate, dextran-coated charcoal). Finally, we have investigated, using the sucrose gradient assay, the nuclear ER presence in 1000g thymic pellet. The results exclude high levels of estrogen-binding proteins while don't suggest their total absence in the female rabbit thymus.     

Within-population variability in a moth sex pheromone blend: genetic basis and behavioural consequences

Evolutionary diversification of sexual communication systems in moths is perplexing because signal and response are under stabilizing selection in many species, and this is expected to constrain evolutionary change. In the moth Heliothis virescens, we consistently found high phenotypic variability in the female sex pheromone blend within each of four geographically distant populations. Here, we assess the heritability, genetic basis and behavioural consequences of this variation. Artificial selection with field-collected moths dramatically increased the relative amount of the saturated compound 16:Ald and decreased its unsaturated counterpart Z11–16:Ald, the major sex pheromone component (high line). In a cross between the high- and low-selected lines, one quantitative trait locus (QTL) explained 11–21% of the phenotypic variance in the 16:Ald/Z11–16:Ald ratio. Because changes in activity of desaturase enzymes could affect this ratio, we measured their expression levels in pheromone glands and mapped desaturase genes onto our linkage map. A delta-11-desaturase had lower expression in females producing less Z11–16:Ald; however, this gene mapped to a different chromosome than the QTL. A model in which the QTL is a trans-acting repressor of delta-11 desaturase expression explains many features of the data. Selection favouring heterozygotes which produce more unsaturated components could maintain a polymorphism at this locus.     

Complex temporal decay: a complete analysis

Relaxation dynamics is universal in science and engineering; its study serves to parameterize a system's response and to help identify a microscopic model of the processes involved. When measured data for a phenomenon cannot be fitted using one exponential, the choice of an alternative function to describe the decay becomes nontrivial. Here, we contrast two different, but fundamentally related approaches to fitting nontrivial decay curves; exponential decomposition and the gamma probability density function. Copyright © 2013 John Wiley & Sons, Ltd.     

Thermococcus kodakarensis Genetics: TK1827-Encoded β-Glycosidase,New Positive-Selection Protocol,and Targeted and Repetitive Deletion Technology

Inactivation of TK1761, the reporter gene established for Thermococcus kodakarensis, revealed the presence of a second β-glycosidase that we have identified as the product of TK1827. This enzyme (pTK1827) has been purified and shown to hydrolyze glucopyranoside but not mannopyranoside, have optimal activity at 95°C and from pH 8 to 9.5, and have a functional half-life of ∼7 min at 100°C. To generate a strain with both TK1761 and TK1827 deleted, a new selection/counterselection protocol has been developed, and the levels of β-glycosidase activity in T. kodakarensis strains with TK1761 and/or TK1827 deleted and with these genes expressed from heterologous promoters are described. Genetic tools and strains have been developed that extend the use of this selection/counterselection procedure to delete any nonessential gene from the T. kodakarensis chromosome. Using this technology, TK0149 was deleted to obtain an agmatine auxotroph that grows on nutrient-rich medium only when agmatine is added. Transformants can therefore be selected rapidly, and replicating plasmids can be maintained in this strain growing in rich medium by complementation of the TK0149 deletion.Members of the Thermococcales, hyperthermophilic Euryarchaea, grow readily under laboratory conditions and are the focus of many basic and applied research projects (2, 8). Their investigation has, however, been limited by the lack of genetics, and it was a seminal advance therefore when Thermococcus kodakarensis (formerly Thermococcus kodakaraensis [1]) was shown to be naturally competent and to recombine added DNA into its genome (21, 23). T. kodakarensis is attractive as an experimental system as a fermentative heterotroph that grows rapidly on a variety of different substrates (1), optimally at 85°C, including substrates from which it generates substantial levels of hydrogen (11). The T. kodakarensis genome sequence and genome microarray assays are established (7, 12, 18) and, since the discovery of transformation (21), inactivation and manipulation of chromosomal genes has revealed novel biochemical pathways, facilitated in vivo evaluations of the archaeal gene expression machinery, and simplified enzyme purifications (3, 5, 6, 9, 10, 12, 15-20, 22-24, 28). For complementation assays and to facilitate heterologous gene expression in T. kodakarensis, shuttle plasmids have been constructed that replicate and confer selectable phenotypes in both T. kodakarensis and Escherichia coli (19), and TK1761 expression has been established as a reporter system that can be used to identify and quantify regulatory elements in T. kodakarensis (18, 20).During the development of the TK1761 reporter system, a T. kodakarensis strain, designated TS416, was constructed with a nonsense mutation in TK1761 (18). This mutation had no discernible effects on growth, confirming that TK1761 was not an essential gene, but lysates of T. kodakarensis TS416 retained a low level β-glycosidase activity. T. kodakarensis apparently, therefore, had a second β-glycosidase, but since this activity was very low and remained constant with changes in TK1761 expression, its presence did not compromise the use of TK1761 expression as a reporter system in the laboratory media used. The existence of a second β-glycosidase did, nevertheless, raise a potential concern for TK1761 assays in cells grown under different conditions in which expression of the gene encoding this second β-glycosidase might not be constant. To address this, we purified and characterized the second β-glycosidase and, to eliminate the concern for the reporter assay, we constructed a T. kodakarensis strain with both TK1761 and the gene (TK1827) that encodes the second β-glycosidase deleted.To construct the double-deletion strain, we developed a new selection/counterselection protocol and have extended this into a procedure that can be used to delete any nonessential gene from the T. kodakarensis genome. A two-gene cassette has been constructed that can be integrated into the T. kodakarensis chromosome at any desired locus by homologous recombination of flanking genes. Expression of the cassette provides a positive selection for transformants and confers sensitivity to 6-methyl purine (6MP^s). Mutants then isolated that are spontaneously resistant to 6-methyl purine (6MP^r) have both the cassette and the adjacent target gene(s) precisely deleted.Sato et al. (21, 23) established the T. kodakarensis transformation protocol by selecting transformants of tryptophan (trpE) and uracil (pyrF) auxotrophs that grew on minimal medium without tryptophan or uracil. Overexpression of the hydroxy-methylglutaryl-coenzyme A reductase encoded by PF1848, cloned from Pyrococcus furiosus, was later found to confer to resistance to simvastatin (15) and mevinolin (19), allowing the selection of T. kodakarensis transformants on rich media that contain either antibiotic. Mutants spontaneously resistant to these antibiotics do, however, occur at experimentally significant frequencies, and these are very expensive reagents for routine use and prohibitively expensive for incorporation into large preparative cultures. With this in mind, to develop an alternative selection that might be used in nutrient-rich media, we used the 6MP cassette system to delete TK0149. As predicted (6), the T. kodakarensis ΔTK0149 strain generated was an agmatine auxotroph that only grows in nutrient-rich media when agmatine is added. When transformed with DNA expressing TK0149, transformants of this strain can be selected directly on standard nutrient-rich media, and complementation of the ΔTK0149 mutation can be used to maintain the presence of an expression plasmid in T. kodakarensis cells grown in large-volume, rich-medium cultures for enzyme purification.     

Oxidised LDL modulate adipogenesis in 3T3-L1 preadipocytes by affecting the balance between cell proliferation and differentiation

The effects of oxidised LDL (oxLDL) on cell proliferation, apoptosis and hormone-induced differentiation have been evaluated for the first time in 3T3-L1 preadipocytes. Unlike control cells, oxLDL-treated preadipocytes showed a high proliferation rate, a low apoptosis level, and an impaired differentiation process with an increased preadipocyte factor-1 (Pref-1) mRNA expression at late times. By silencing Pref-1 mRNA or inhibiting its expression with an increased dexamethasone concentration, differentiation occurred as usual, which demonstrates the key role of Pref-1 overexpression. The results suggest a specific action of oxLDL on the adipogenesis inhibitor Pref-1, as indicated also by its reappearance in mature adipocytes treated with oxLDL. The inhibitory effects of oxLDL on differentiation required oxLDL uptake by CD36, and were associated with lipoprotein lipids. These results point to oxLDL as a modulator of adipose tissue mass and as possible link between obesity and its clinical complications.