Further Studies on the Ribosomal RNA Cistrons of SCIARA COPROPHILA (Diptera)

Additional experiments with homologous as well as heterologous hybridization confirmed our previous finding in Sciara coprophila that XX females have nearly twice the number of ribosomal RNA cistrons as XO males. A comparison between two different X' chromosomes revealed that only the one carrying the irradiation-induced Wavy mutation has a deletion of 70% of its ribosomal RNA cistrons as compared to the standard X. The deletion is relatively stable, and the remaining ribosomal RNA cistrons donot appear to undergo disproportionate replication or magnification as in Drosophila. Homologous hybridization experiments revealed an unusually low reiteration of ribosomal RNA cistrons in this fly, 45 gene copies per X chromosome. The question is raised as to whether such a low number of cistrons may be related to the unusual nucleolar condition encountered in the Sciaridae.     

Modulation by prostaglandins of contractions in guinea-pig ileum

A high concentration of indomethacin (40μg/ml) substantially reduced contractions of guinea-pig isolated ileum in Krebs solution to nerve stimulation with electrical pulses or nicotine. Responses to acetylcholine and histamine were also inhibited, but to a smaller extent. Low concentrations of prostaglandin E₂ (2 or 4ng/ml) mainly restored all the excitatory responses. Using a modified bathing solution (lacking in phosphate and with some other changes) indomethacin 0.36μg/ml selectively inhibited nerve-mediated contractions. The results explain differences in various reports, and support the possibility that prostaglandins modulate the response to cholinergic nerve activity.     

Optimising Immunogenicity with Viral Vectors: Mixing MVA and HAdV-5 Expressing the Mycobacterial Antigen Ag85A in a Single Injection

The Bacillus Calmette - Guerin (BCG) vaccine provides a critical but limited defense against Mycobacterium tuberculosis (M.tb). More than 60 years after the widespread introduction of BCG, there is an urgent need for a better vaccine. A large body of pre-clinical research continues to support ongoing clinical trials to assess whether viral vectors expressing M.tb antigens that are shared by BCG and M.tb, can be used alongside BCG to enhance protection. A major focus involves using multiple unique viral vectors to limit anti-vector immunity and thereby enhance responses to the insert antigen delivered. The successful introduction of viral vector vaccines to target M.tb and other pathogens will be reliant on reducing the costs when using multiple vectors and inhibiting the development of unwanted anti-vector responses that interfere with the response to insert antigen. This study examines methods to reduce the logistical costs of vaccination by mixing different viral vectors that share the same insert antigen in one vaccine; and whether combining different viral vectors reduces anti-vector immunity to improve immunogenicity to the insert antigen. Here we show that a homologous prime-boost regimen with a mixture of MVA (Modified Vaccinia virus Ankara) and Ad5 (human adenovirus type 5) vectors both expressing Ag85A in a single vaccine preparation is able to reduce anti-vector immunity, compared with a homologous prime-boost regimen with either vector alone. However, the level of immunogenicity induced by the homologous mixture remained comparable to that induced with single viral vectors and was less immunogenic than a heterologous Ad5 prime-MVA-boost regimen. These findings advance the understanding of how anti-vector immunity maybe reduced in viral vector vaccination regimens. Furthermore, an insight is provided to the impact on vaccine immunogenicity from altering vaccination methods to reduce the logistical demands of using separate vaccine preparations in the field.     

Nicotinic Receptor-Mediated Release of Noradrenaline in the Human Neuroblastoma SH-SY5Y

Abstract: Dimethylphenylpiperazinium iodide (a nicotinic agonist) evokes noradrenaline release from human neuroblastoma SH-SY5Y cells that have been pretreated with 12- O -tetradecanoylphorbol 13-acetate for 8 min. This effect of dimethylphenylpiperazinium iodide was inhibited by 1 μ M mecamylamine but not by 1 μ M atropine, which suggests that SH-SY5Y cells express nicotinic receptors coupled to the release of noradrenaline. Dimethylphenylpiperazinium iodide-evoked release was enhanced by 5 μ M Bay K 8644 (an L-type calcium agonist) and inhibited by 1 μ M nifedipine. Dimethylphenylpiperazinium iodide depolarised SH-SY5Y cells and enhanced the level of intracellular calcium in cells loaded with fura 2. The effects of dimethylphenylpiperazinium iodide on noradrenaline release, depolarisation, and intracellular calcium levels were all inhibited by 1 μ M desmethylimipramine. The results of this study show that nicotinic receptors in SH-SY5Y cells stimulate noradrenaline release by activation of L-type calcium channels.     

A Novel Piggybac Transposon Inducible Expression System Identifies a Role for Akt Signalling in Primordial Germ Cell Migration

In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC) lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development.     

Defective ion regulation in a class of membrane-excitation mutants in Paramecium

The “paranoiac” mutants of Paramecium aurelia show prolonged backward swimming in solutions containing Na⁺, unlike wild-type paramecia, which jerk back and forth in Na⁺ solutions. The paranoiac mutants in Na⁺ solutions also show large losses of cellular K⁺ and large influxes of Na⁺. Three different paranoiac mutants all show similar defects in ion regulation but to different degrees. Wild-type Paramecium, in contrast, shows no Na⁺-dependent loss of cellular K⁺ and a much smaller Na⁺ influx. In K⁺-containing solutions, there is no difference between wild-type and paranoiac paramecia with respect to their cellular K⁺ content.The Na⁺ influx, the K⁺ loss, and the duration of backward swimming are all proportional to the extracellular Na⁺ concentration. Electrophysiologically, the backward swimming of the paranoiac mutants corresponds to a prolonged depolarization of the membrane potential, while the backward jerks of wild-type Paramecium correspond to a series of transient depolarizations. We propose that the large Na⁺ influxes and the large K⁺ effluxes in paranoiacs occur during the periods of backward swimming, while the membrane is depolarized.