Global down-regulation of gene expression in the brain using RNA interference, with emphasis on monoamine transporters and GPCRs: implications for target characterization in psychiatric and neurological disorders

RNA interference (RNAi) is a natural mechanism for regulating gene expression, which exists in plants, invertebrates, and mammals. We investigated whether non-viral infusion of short interfering RNA (siRNA) by the intracerebroventricular route would enable a sequence-specific gene knockdown in the mouse brain and whether the knockdown translates into disease-relevant behavioral changes. Initially, we targeted enhanced green fluorescent protein (EGFP) in mice overexpressing EGFP. A selective knockdown of both EGFP protein and mRNA was observed throughout the brain, with lesser down-regulation in regions distal to the infusion site. We then targeted endogenous genes, encoding the dopamine (DAT) and serotonin transporters (SERT). DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein and elicited hyperlocomotion similar, but delayed, to that produced on infusion of GBR-12909, a potent and selective DAT inhibitor. Similarly, SERT-siRNA infusion resulted in significant knockdown of SERT mRNA and protein and elicited reduced immobility in the forced swim test similar to that obtained on infusion of citalopram, a very selective and potent SSRI. Application of this non-viral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.     

Field performance of Solanum sisymbriifolium, a trap crop for potato cyst nematodes. II. Root characteristics

Hatching of potato cyst nematodes is induced by root exudates of Solanaceae, such as Solanum sisymbriifolium, and is therefore related to root length distribution of this crop. A mathematical model was derived to relate the hatching potential to root length density (RLD). A series of field experiments was carried out to study actual root length distribution of S. sisymbriifolium in relation to shoot properties and to provide input into the model. Using a modified Poisson distribution formula for the three‐dimensional distribution of roots in a volume of soil, the relation between the zone of influence of hatching agents and the RLD could be derived. On this basis, the minimal RLD was estimated, which is needed to expose 75%, 90% or 95% of cysts to root exudates, as a function of the length of the zone of influence of hatching agents on cysts. The logarithm of the total root length showed a linear relation with the logarithms of above‐ground biomass and with leaf area index. Root diameter distribution was the same for all crops examined and independent of soil depth. Fine roots (<0.4 mm in diameter) constituted around 50% of total root length. Using a zone of influence of 1.00, 0.75 and 0.50 cm around the centre of each root, a minimal RLD for sufficient soil exploration (75%) was estimated. Depth at which that minimal RLD was exceeded was linearly related to total root length (km m^?2) and to above‐ground crop biomass, enabling estimations being made of the potential hatching efficacy as related to measurable properties of S. sisymbriifolium crops. The proposed approach to derive potential hatching effects from crop properties needs further validation; particularly, the distance of influence of root exudates is a critical factor.     

THE EFFICIENCY OF THE ASSAY FOR HAEMOPIETIC COLONY FORMING CELLS

The quantitative efficiency of the spleen colony assay in mice is discussed in the light of recent findings on the kinetics of colony forming cells. Arguments are presented showing that the f factor, the 2 hr CFU recovery fraction in the spleen, markedly over-estimates the assay efficiency which is the ratio of the numbers of colony forming units and colony forming cells.     

Systemic deletion of the myelin-associated outgrowth inhibitor Nogo-A improves regenerative and plastic responses after spinal cord injury

To investigate the role of the myelin-associated protein Nogo-A on axon sprouting and regeneration in the adult central nervous system (CNS), we generated Nogo-A-deficient mice. Nogo-A knockout (KO) mice were viable, fertile, and not obviously afflicted by major developmental or neurological disturbances. The shorter splice form Nogo-B was strongly upregulated in the CNS. The inhibitory effect of spinal cord extract for growing neurites was decreased in the KO mice. Two weeks following adult dorsal hemisection of the thoracic spinal cord, Nogo-A KO mice displayed more corticospinal tract (CST) fibers growing toward and into the lesion compared to their wild-type littermates. CST fibers caudal to the lesion-regenerating and/or sprouting from spared intact fibers-were also found to be more frequent in Nogo-A-deficient animals.     

Induction of SM-alpha-actin expression by mechanical strain in adult vascular smooth muscle cells is mediated through activation of JNK and p38 MAP kinase

Mechanical forces have direct effects on the growth and differentiation of vascular smooth muscle. The goal of this study was to examine the effects of cyclic mechanical strain on expression of smooth muscle-alpha-actin (SM-alpha-actin), a marker for the differentiated state of vascular smooth muscle, in cultured rat aortic smooth muscle cells (VSMC). Cells grown on dishes coated with either laminin or pronectin were subjected to mechanical strain and effects on expression of SM-alpha-actin were evaluated using the Flexercell Strain Unit. Application of mechanical strain to cells in full media increased SM-alpha-actin protein expression and promoter activity. This was not associated with any effect on growth. Mechanical strain increased activity of all three members of the MAP kinase family (ERKs, JNKs, and p38 MAP kinase), with similar kinetics. Inhibition of either JNKs or p38 MAP kinase blocked the strain-induced increase in SM-alpha-actin promoter activity, and expression of constitutively active forms of JNK or MKK6, a p38 kinase, increased promoter activity. These studies indicate that in adult VSMC, mechanical strain leads to increased expression of smooth muscle markers, resulting in a more contractile phenotype.     

Generation of Campylobacter jejuni genetic diversity in vivo

Molecular epidemiology studies suggest that horizontal genetic exchange is a major cause of pathogen biodiversity. We tested this concept for the bacterial enteropathogen Campylobacter jejuni by seeking direct in vivo evidence for the exchange of genetic material among Campylobacter strains. For this purpose, two antibiotic resistance markers were inserted into the hipO or htrA gene of genetically distinct and naturally transformable C. jejuni strains. Genetic exchange of the resistance markers was analysed after co-cultivation of homologous and heterologous strains in vitro and in vivo during experimental infection of chickens. Double-resistant recombinants were obtained both in vitro and from the chicken intestine for all combinations of strains tested. Bidirectional genetic exchange of DNA between homologous and heterologous strains was confirmed by Southern blotting in combination with flaA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoresis (PFGE). Extensive PFGE analyses of isolated recombinants indicated the frequent occurrence of genetic rearrangements during the experimental infection, in addition to the homologous recombination of the antibiotic resistance genes. Together, the data indicate unequivocally that interstrain genetic exchange as well as intragenomic alterations do occur in vivo during C. jejuni infection. These events probably explain the genome plasticity observed for this pathogen.     

Plant–soil biota interactions and spatial distribution of black cherry in its native and invasive ranges

One explanation for the higher abundance of invasive species in their non‐native than native ranges is the escape from natural enemies. But there are few experimental studies comparing the parallel impact of enemies (or competitors and mutualists) on a plant species in its native and invaded ranges, and release from soil pathogens has been rarely investigated. Here we present evidence showing that the invasion of black cherry (Prunus serotina) into north‐western Europe is facilitated by the soil community. In the native range in the USA, the soil community that develops near black cherry inhibits the establishment of neighbouring conspecifics and reduces seedling performance in the greenhouse. In contrast, in the non‐native range, black cherry readily establishes in close proximity to conspecifics, and the soil community enhances the growth of its seedlings. Understanding the effects of soil organisms on plant abundance will improve our ability to predict and counteract plant invasions.     

Induction of smooth muscle alpha-actin in vascular smooth muscle cells by arginine vasopressin is mediated by c-Jun amino-terminal kinases and p38 mitogen-activated protein kinase

Exposure of vascular smooth muscle cells to arginine vasopressin (AVP) increases smooth muscle alpha-actin (SM-alpha-actin) expression through activation of the SM- alpha-actin promoter. The goal of this study was to determine the role of the mitogen-activated protein kinase (MAP kinase) family in regulation of SM-alpha-actin expression. AVP activated all three MAP kinase family members: ERKs, JNKs, and p38 MAP kinase. Inhibition of JNKs or p38 decreased AVP-stimulated SM-alpha-actin promoter activity, whereas inhibition of ERKs had no effect. A 150-base pair region of the promoter containing two CArG boxes was sufficient to mediate regulation by vasoconstrictors. Mutations in either CArG box decreased AVP-stimulated promoter activity. Electrophoretic mobility shift assays using oligonucleotides corresponding to either CArG box resulted in a complex of similar mobility whose intensity was increased by AVP. Antibodies against serum response factor (SRF) completely super-shifted this complex, indicating that SRF binds to both CArG boxes. Overexpression of SRF increased basal promoter activity, but activity was still stimulated by AVP. AVP stimulation rapidly increased SRF phosphorylation. These data indicate that both JNKs and p38 participate in regulation of SM- alpha-actin expression. SRF, which binds to two critical CArG boxes in the promoter, represents a potential target of these kinases.     

Host-related genetic differentiation in the anther smut fungus Microbotryum violaceum in sympatric, parapatric and allopatric populations of two host species Silene latifolia and S. dioica

We investigated genetic diversity in West European populations of the fungal pathogen Microbotryum violaceum in sympatric, parapatric and allopatric populations of the host species Silene latifolia and S. dioica, using four polymorphic microsatellite loci. In allopatric host populations, the fungus was highly differentiated by host species, exhibiting high values of F(ST) and R(ST), and revealed clear and distinct host races. In sympatric and parapatric populations we found significant population differentiation as well, except for one sympatric population in which the two host species grew truly intermingled. The mean number of alleles per locus for isolates from each of the host species was significantly higher in sympatric/parapatric than in allopatric populations. This suggests that either gene flow between host races in sympatry, or in case of less neutral loci, selection in a more heterogeneous host environment can increase the level of genetic variation in each of the demes. The observed pattern of host-related genetic differentiation among these geographically spread populations suggest a long-term divergence between these host races. In sympatric host populations, both host races presumably come in secondary contact, and host-specific alleles are exchanged depending on the amount of fungal gene flow.