Differential expression in male and female mouse liver of very similar mRNAs specified by two group 1 major urinary protein genes.

The major urinary proteins of the mouse are encoded by a large multigene family composed of several distinct groups of genes distinguished by differences in sequence and expression characteristics. The genes in the largest group (group 1) show greater than 99% pairwise similarity in their exons. By hybridization between RNA and a specifically designed oligonucleotide, we confirmed that genes of this group are expressed mainly in the liver. By using additional gene-specific oligonucleotide probes, we have been able to distinguish between the species of mRNA corresponding to two of these genes and to measure their abundance in male and female liver. Both mRNAs are present in male liver at high but different levels. Both are also present in female liver, one at a much lower level than in the male and the second at a very low level indeed. Both are present at male levels in the livers of females induced with testosterone. These results show unequivocally that the expression of different group 1 Mup genes is differentially influenced by the hormonal status of the mouse.     

In vitro propagation of cassava (Manihot esculenta Crantz)

A method is presented for the rapid in vitro propagation of cassava (Manihot esculenta Crantz). Nodal explants were induced to grow as multiple-shoot cultures on a medium containing 1.0 M 6-benzylamino purine (BAP), supplemented with 0.25 M -naphthaleneacetic acid (NAA). Nodes were removed from the shoots after three weeks of growth and subcultured on fresh culture medium. An average of 7.0 nodes were produced from each explanted node after three weeks in culture. Nodal explants were transferred to a medium containing 2.5 M indole-3-butyric acid (IBA) to improve root initiation on the developing plantlets. Plant establishment was possible upon transfer to soil. In vitro propagation offers enhanced rates of multiplication over more conventional methods of propagation. In addition, in vitro propagation facilitates the storage and international exchange of cassava germplasm.     

Compensation in pre-synaptic dopaminergic function following nigrostriatal damage in primates

Clinical symptoms of Parkinson's disease only become evident after 70-80% reductions in striatal dopamine. To investigate the importance of pre-synaptic dopaminergic mechanisms in this compensation, we determined the effect of nigrostriatal damage on dopaminergic markers and function in primates. MPTP treatment resulted in a graded dopamine loss with moderate to severe declines in ventromedial striatum (approximately 60-95%) and the greatest reductions (approximately 95-99%) in dorsolateral striatum. A somewhat less severe pattern of loss was observed for striatal nicotinic receptor, tyrosine hydroxylase and vesicular monoamine transporter expression. Declines in striatal dopamine uptake and transporter sites were also less severe than the reduction in dopamine levels, with enhanced dopamine turnover in the dorsolateral striatum after lesioning. The greatest degree of adaptation occurred for nicotine-evoked [(3)H]dopamine release from striatal synaptosomes, which was relatively intact in ventromedial striatum after lesioning, despite > 50% declines in dopamine. This maintenance of evoked release was not due to compensatory alterations in nicotinic receptor characteristics. Rather, there appeared to be a generalized preservation of release processes in ventromedial striatum, with K(+)-evoked release also near control levels after lesioning. These combined compensatory mechanisms help explain the finding that Parkinson's disease symptomatology develops only with major losses of striatal dopamine.     

Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization

Background

Pulmonary-delivered gene therapy promises to mitigate vaccine safety issues and reduce the need for needles and skilled personnel to use them. While plasmid DNA (pDNA) offers a rapid route to vaccine production without side effects or reliance on cold chain storage, its delivery to the lung has proved challenging. Conventional methods, including jet and ultrasonic nebulizers, fail to deliver large biomolecules like pDNA intact due to the shear and cavitational stresses present during nebulization.

Methods

In vitro structural analysis followed by in vivo protein expression studies served in assessing the integrity of the pDNA subjected to surface acoustic wave (SAW) nebulisation. In vivo immunization trials were then carried out in rats using SAW nebulized pDNA (influenza A, human hemagglutinin H1N1) condensate delivered via intratracheal instillation. Finally, in vivo pulmonary vaccinations using pDNA for influenza was nebulized and delivered via a respirator to sheep.

Results

The SAW nebulizer was effective at generating pDNA aerosols with sizes optimal for deep lung delivery. Successful gene expression was observed in mouse lung epithelial cells, when SAW-nebulized pDNA was delivered to male Swiss mice via intratracheal instillation. Effective systemic and mucosal antibody responses were found in rats via post-nebulized, condensed fluid instillation. Significantly, we demonstrated the suitability of the SAW nebulizer to administer unprotected pDNA encoding an influenza A virus surface glycoprotein to respirated sheep via aerosolized inhalation.

Conclusion

Given the difficulty of inducing functional antibody responses for DNA vaccination in large animals, we report here the first instance of successful aerosolized inhalation delivery of a pDNA vaccine in a large animal model relevant to human lung development, structure, physiology, and disease, using a novel, low-power (<1 W) surface acoustic wave (SAW) hand-held nebulizer to produce droplets of pDNA with a size range suitable for delivery to the lower respiratory airways.     

Comparative <Emphasis Type="Italic">in vivo</Emphasis> infection models yield insights on early host immune response to Campylobacter in chickens

Salmonella typhimurium and Campylobacter jejuni pose significant risks to human health and poultry are a major vector for infection. Comparative in vivo infection models were performed to compare the avian host immune response to both bacterial species. Forty-five commercial broiler chickens were orally challenged with either C. jejuni or S. typhimurium whilst 60 similar control birds were mock challenged in parallel. Birds were sacrificed at 0, 6, 20 and 48 h post-infection and cloacal swabs, blood and tissue samples taken. Peripheral blood leukocytes were isolated for flow cytometric analyses and RNA was extracted for gene expression profiling. Colonisation patterns were markedly different between the two bacterial species, with systemic colonisation of Campylobacter outside the gastrointestinal tract. Salmonella infection induced significant changes in circulating heterophil and monocyte/macrophage populations, whilst Campylobacter infection had no effect on the heterophil numbers but caused a significant early increase in circulating monocytes/macrophages. Toll-like receptor 1 (TLR1) gene expression was decreased, and avian β-defensin (AvBD) gene expression (AvBD3, AvBD10 and AvBD12) was significantly increased in response to Salmonella infection (P < 0.05). In contrast, Campylobacter infection induced increased TLR21 gene expression but significantly reduced expression of seven antimicrobial peptide (AMP) genes (AvBD3, AvBD4, AvBD8, AvBD13, AvBD14, CTHL2 and CTHL3; P < 0.05). Considered together, microbiological, cellular and gene expression profiles indicate that the innate immune system responds differently to Salmonella and to Campylobacter infection. Furthermore, reduction in the expression of AMPs may play a role in the persistence of high level colonisation of the host by Campylobacter. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Low genetic diversity contrasts with high phenotypic variability in heptaploid Spartina densiflora populations invading the Pacific coast of North America

Species can respond to environmental pressures through genetic and epigenetic changes and through phenotypic plasticity, but few studies have evaluated the relationships between genetic differentiation and phenotypic plasticity of plant species along changing environmental conditions throughout wide latitudinal ranges. We studied inter‐ and intrapopulation genetic diversity (using simple sequence repeats and chloroplast DNA sequencing) and inter‐ and intrapopulation phenotypic variability of 33 plant traits (using field and common‐garden measurements) for five populations of the invasive cordgrass Spartina densiflora Brongn. along the Pacific coast of North America from San Francisco Bay to Vancouver Island. Studied populations showed very low genetic diversity, high levels of phenotypic variability when growing in contrasted environments and high intrapopulation phenotypic variability for many plant traits. This intrapopulation phenotypic variability was especially high, irrespective of environmental conditions, for those traits showing also high phenotypic plasticity. Within‐population variation represented 84% of the total genetic variation coinciding with certain individual plants keeping consistent responses for three plant traits (chlorophyll b and carotenoid contents, and dead shoot biomass) in the field and in common‐garden conditions. These populations have most likely undergone genetic bottleneck since their introduction from South America; multiple introductions are unknown but possible as the population from Vancouver Island was the most recent and one of the most genetically diverse. S. densiflora appears as a species that would not be very affected itself by climate change and sea‐level rise as it can disperse, establish, and acclimate to contrasted environments along wide latitudinal ranges.     

Identification of a microtubule-based cytoplasmic motor in the nematode C. elegans

C. elegans contains a microtubule binding protein that resembles both dynein and kinesin. This protein has a MgATPase activity and copurifies on both sucrose gradients and DEAE Sephadex columns with a polypeptide of Mr approximately 400 kd. The ATPase activity is 50% inhibited by 10 microM vanadate, 1 mM N-ethyl maleimide, or 5 mM AMP-PNP; it is enhanced 50% by 0.2% Triton. The 400 kd polypeptide is cleaved at a single site by ultraviolet light in the presence of ATP and vanadate. In these ways, the protein resembles dynein. The protein also promotes ATP-dependent translocation of microtubules or axonemes, "plus" ends trailing. This property is kinesin-like; however, the motility is blocked by 5 microM vanadate, 1 mM N-ethyl maleimide, 0.5 mM ATP-gamma-S, or by ATP-vanadate-UV cleavage of the 400 kd polypeptide, characteristics that differ from kinesin. We propose that this protein is a novel microtubule translocator.     

Characterization of Acetylcholine-induced Increases in Cytosolic Free Calcium Concentration in Individual Rat Pancreatic β-cells

The intracellular free Ca²⁺ ion concentration ([Ca²⁺]i) was measured using fura-2 microspec-trofluorimetry in individual rat pancreatic β-cells prepared by enzymatic digestion and fluorescence-activated cell sorting. The mean basal concentration of [Ca²⁺]i in β-cells in the presence of 4.4 mM glucose and 1.8 mM Ca²⁺ was 112±1.6 nM (n=207). The action of acetylcholine (ACh) was concentration-dependent, and raising the concentration resulted in [Ca²⁺]i spikes of increasing amplitude and duration in some, but not all of the β-cells. In addition, the β-cells demonstrated variable sensitivity to ACh. The increases in [Ca²⁺]i were rapid, transient and were blocked by atropine at 10^-6M. A brief exposure to 50 mM K⁺ resulted in a transient increase in [Ca²⁺]i similar to that induced by ACh, but resistant to atropine. A high concentration of ACh (100μL 10^-4M or 10^-3M) induced [Ca²⁺]i oscillations in 11 out of 57 β-cells in the presence of 4.4 mM glucose. Using calcium channel blockers and Ca²⁺ free medium, the source of the increase in [Ca²⁺]i was deduced to be from extracellular spaces. Changing the temperature from 22 to 37°C did not affect the action of ACh on [Ca²⁺]i. These data strongly suggest that ACh exerted a direct action on [Ca²⁺]i in normal rat pancreatic β-cells and support a role for Ca²⁺ as a second messenger in the action of ACh.     

Interactions between fish,grazing invertebrates and algae in a New Zealand stream: a trophic cascade mediated by fish-induced changes to grazer behaviour?

Experiments in laboratory stream channels compared the behaviour of Deleatidium mayfly nymphs in the absence of fish with that in the presence of either native common river galaxias (Galaxias vulgaris Stokell) or introduced brown trout (Salmo trutta L.). Galaxias present similar predation risks to prey during day and night but are more active at night. Whereas, trout present a higher predation risk during the day. Deleatidium maintained a fixed nocturnal drift periodicity that is characteristic of streams containing visually feeding fish regardless of the nature of the predation regime presented in the laboratory. However, the number on the substratum surface, and therefore able to graze algae, was lower when fish were present than when they were absent. The number was lower during the day in the presence of trout, when they present the highest predation risk, and lower during the night compared to the day in trials with galaxias when galaxias activity disturbs Deleatidium from the substratum. Increases in the probability of Deleatidium leaving a patch, reductions in the proportion of mayflies on high quality patches and reductions in the distance travelled from refuge also reflected variations in the predation regime. Similar differences in positioning were observed under the same predation regimes in in situ channels in the Shag River and these were associated with differences in algal biomass. Algal ash-free dry mass (AFDM) and chlorophyll a (chl a) were higher on the tops of cobbles when fish were present. Fish also affected the biomass and the distribution of algae on cobbles as AFDM and chl a were higher on the sides of cobbles from channels with trout compared to those with galaxias. Changes in grazing behaviour, caused by predator avoidance, are likely to have been responsible for differences in algal biomass because no significant differences were detected between treatments in the biomass of Deleatidium or of total invertebrates.