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Neuroimaging studies in post-traumatic stress disorder.

The authors review some of the advances that have been made in understanding the structural, biochemical, and functional neuroanatomy of post-traumatic stress disorder (PTSD). First, the authors review the primary brain regions that had been hypothesized a priori, from the phenomenology and neurobiology of PTSD, to be implicated in the pathophysiology. Next, they review findings from neuroimaging studies of these brain regions in PTSD, and explain the various experimental methods and imaging technologies used in these studies. A broader perspective, including a discussion of additional brain areas that may be involved in PTSD, is synthesized. The authors conclude with a rationale and approach for studies testing sharply defined hypotheses and those using multidisciplinary strategies that integrate neuroimaging data with other cognitive, biologic, and genetic tools to study this complex disorder.

Amygdala↗

Autonomic dysreflexia in a mouse model of spinal cord injury.

Most experimental studies of spinal cord injury have centered on the rat as an experimental model. A shift toward a mouse model has occurred in recent years because of its usefulness as a genetic tool. While many studies have concentrated on motor function and the inflammatory response following spinal cord injury in the mouse, the development of autonomic dysreflexia after injury has yet to be described. Autonomic dysreflexia is a condition in which episodic hypertension develops after injuries above the mid-thoracic segment of the spinal cord. In this study 129Sv mice received a spinal cord transection at the second thoracic segment. The presence of autonomic dysreflexia was assessed 2 weeks later. Blood pressure responses to stimulation were as follows: moderate cutaneous pinch caudal to the injury (35+/-6 mm Hg), tail pinch (25+/-7 mm Hg), and a 0.3 ml balloon distension of the colon (37+/-4 mm Hg). Previous reports have suggested that small diameter primary afferent fiber sprouting after spinal cord injury may be responsible for the development of autonomic dysreflexia. In order to determine whether autonomic dysreflexia in the mouse may be caused by a similar mechanism, the size of the small diameter primary afferent arbor in spinal cord-injured and sham-operated animals was assessed by measuring the area occupied by calcitonin gene-related peptide-immunoreactive fibers. The percentage increase in the area of the small diameter primary afferent arbor in transected over sham-operated spinal cords was 46%, 45% and 80% at spinal segments thoracic T5-8, thoracic T9-12 and thoracic T13-lumbar L2 respectively. This study demonstrates the development of autonomic dysfunction in a mouse model of spinal cord injury that is associated with sprouting of calcitonin gene-related peptide fibers. These results provide strong support for the use of this mouse model of spinal cord injury in the study of autonomic dysreflexia.

Animals↗

Experimental studies of adult Drosophila chemosensory behaviour.

Drosophila melanogaster is a powerful animal model to study the processes underlying behavioural responses to chemical cues. This paper provides a review of the important literature to present recent advances in our understanding of how gustatory and olfactory stimuli are perceived. An overview is given of the experimental procedures currently used to characterize the fly chemosensory behaviour. Since this species provides extremely useful genetic tools, a focus is made on those allowing to manipulate behaviour, and hence to understand its molecular and cellular bases. Such tools include single-gene mutants and the Gal4/UAS system. They can be combined with studies of the natural polymorphism of behavioural responses. Recent data obtained with these various approaches unravel some important aspects of taste and olfaction. These appear as rather complex processes, as revealed by results showing dose-dependence, plasticity and sexual dimorphism. Taken together, these results and the available tools open interesting perspectives for the years to come, in our attempts to make the link between genes and behaviour.

Journal Article↗

A novel technique for inducing local inner ear damage.

With significant development of mouse genomics and the availability of transgenic and knockout mice, the mouse will be the preferred animal model for inner ear research. However, few studies have used mice as experimental animals for examination of hair cell degeneration, because of their relative resistance to ototoxic agents and difficulties in surgical treatment. This study presents a model for induction of apoptotic cell death in sensory epithelia of the mouse inner ear using injection of neomycin into the posterior semicircular canal. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay revealed that local application of neomycin produced sufficient induction of apoptotic cell death in both auditory and vestibular epithelia over a definite time course. Supplementation of the general caspase-inhibitor significantly reduced induction of TUNEL-positive cells, indicating caspase-dependency of apoptotic cell death observed in the present model. In addition, the approach to the posterior semicircular canal was an easy technique, and sham-operation induced no significant induction of TUNEL-positive cells. This model, hence, enables the use of various genetic tools in studies for mechanisms of hair cell apoptosis.

Amino Acid Chloromethyl Ketones↗

Influenza virus genetics.

Significant progress has been made in understanding the process of influenza A virus replication in cell culture; however, much less is known about the genetic control of virus-host interactions in disease. This review provides an overview of the genetic analysis of influenza virus biology. The functional map of the individual genes of influenza A virus is presented as well as the status of our current understanding of pathogenesis. Influenza has a segmented genome so it is possible to obtain reassortants that contain novel combinations of genome segments derived from different viruses. This is a very useful genetic tool and is also an important aspect of influenza evolution and biology. Human influenza viruses originate from avian strains of influenza virus so that influenza infection is at its basis a zoonosis. Influenza virus strains are host-restricted, however, and avian strains must be adapted to the human host. So questions of host-range and interaction with host factors are important determinants of the ability of influenza virus to cause disease in humans. Host-range is restricted primarily due to host-specific interactions of the ribonucleocapsid and the viral receptor. There are two classes of drugs for inhibiting influenza infection, amantadine HCl and neuraminidase inhibitors. The mode of action and basis for resistance to these drugs are presented. Prospective targets for antiviral therapy are also discussed.

Amantadine↗

Genetic studies of a thermoregulated gene in the psychrotrophic bacterium Pseudomonas fluorescens.

In the psychrotrophic bacterium Pseudomonas fluorescens, some genes are thermoregulated: they are maximally expressed at a particular temperature within the broad range of temperatures that allow growth of this bacterium. To study this regulation, random transcriptional insertion fusions were obtained by means of mini-Tn5lacZ1 or mini-Tn5luxAB transposition. One fusion was studied in which beta-galactosidase production was maximal at a low-growth temperature. The mutated gene (that we call xsf) was highly homologous to xseA from Escherichia coli (and from other bacteria) which encodes the large subunit of exonuclease VII. Genetic tools were constructed in order to analyse and manipulate this fusion: a plasmid derived from R68.45 was used for chromosome transfer and a replacement vector was constructed to allow in situ marker exchange of the mini-Tn5lacZ1 by an Hg(r) interposon. This vector was used to make double mutants and hence to study the effect of the insertion in xsf on the expression of other fusions. Six genes were thereby identified with a decreased expression in an xsf- background and with different characteristics of thermoregulation.

Artificial Gene Fusion↗

A closer look at proteolysis and MHC-class-II-restricted antigen presentation.

Antigen presentation by MHC class II molecules relies on the action of endocytic proteases, which are differentially expressed in antigen-presenting cells and are regulated by different components of the immune system. Endocytic enzymes process and convert exogenous antigens into peptidic determinants capable of interaction with MHC class II molecules. Chemical and genetic tools have recently been developed to study the role of lysosomal proteases in antigen presentation.

Animals↗

Modern methods for probing RNA structure.

Molecular biologists have been remarkably successful in dividing large RNAs into small functional modules manageable for NMR and X-ray studies. At the same time biophysical, biochemical and genetic tools in RNA structure determination have reached a level of sophistication, at which we start to see a glimpse of molecular dynamics and the mechanism of RNA mediated catalysis.

Biochemistry↗

Olfaction in Drosophila.

The fruit fly, Drosophila melanogaster, is equipped with a sophisticated olfactory sensory system that permits it to recognize and discriminate hundreds of discrete odorants. The perception of these odorants is essential for the animal to identify relevant food sources and suitable sites for egg-laying. Advances in the last year have begun to define the molecular basis of this insect's discriminatory power. The identification of a large multi-gene family of candidate Drosophila odorant receptors suggests that, as in other animals, a multitude of distinct odorants is recognized by a diversity of ligand-binding receptors. How olfactory signals are transduced and interpreted by the brain remains an important question for future analysis. The availability of genetic tools and a complete genome sequence makes Drosophila a particularly attractive organism for studying the molecular basis of olfaction.

Animals↗

Optimising enzyme function by directed evolution.

The past decade has seen a revolution in our ability to engineer designer enzymes using genetic tools that mimic evolution on a laboratory timescale. Many excellent examples of directed evolution applied to a wide range of enzymes have clearly demonstrated its future role in adapting enzymes for use in the chemical industry. Recent advances in 'smart' library design and computational screening are now permitting much deeper searches of sequence space, which potentially increases the extent to which enzyme function can be modified.

Animals↗

Molecular genetic approaches to the targeted suppression of neuronal activity.

Understanding how the diverse cells of the nervous system generate sensations, memories and behaviors is a profound challenge. This is because the activity of most neurons cannot easily be monitored or individually manipulated in vivo. As a result, it has been difficult to determine how different neurons contribute to nervous system function, even in simple organisms like Drosophila. Recent advances promise to change this situation by supplying molecular genetic tools for modulating neuronal activity that can be deployed in a spatially and temporally restricted fashion. In some cases, targeted groups of neurons can be 'switched off' and back 'on' at will in living, behaving animals.

Animals↗

Genetically expressed cameleon in Drosophila melanogaster is used to visualize olfactory information in projection neurons.

Complex external stimuli such as odorants are believed to be internally represented in the brain by spatiotemporal activity patterns of extensive neuronal ensembles. These activity patterns can be recorded by optical imaging techniques. However, optical imaging with conventional fluorescence dyes usually does not allow for resolving the activity of biologically defined groups of neurons. Therefore, specifically targeting reporter molecules to neuron populations of common genetic identity is an important goal. We report the use of the genetically encoded calcium-sensitive fluorescence protein cameleon 2.1 in the Drosophila brain. We visualized odorant-evoked intracellular calcium concentration changes in selectively labeled olfactory projection neurons both postsynaptically in the antennal lobe, the primary olfactory neuropil, and presynaptically in the mushroom body calyx, a structure involved in olfactory learning and memory. As a technical achievement, we show that calcium imaging with a genetically encoded fluorescence probe is feasible in a brain in vivo. This will allow one to combine Drosophila's advanced genetic tools with the physiological analysis of brain function. Moreover, we report for the first time optical imaging recordings in synaptic regions of the Drosophila mushroom body calyx and antennal lobe. This provides an important step for the use of Drosophila as a model system in olfaction.

Animals↗

Composition and functional characterization of yeast 66S ribosome assembly intermediates.

The pathway and complete collection of factors that orchestrate ribosome assembly are not clear. To address these problems, we affinity purified yeast preribosomal particles containing the nucleolar protein Nop7p and developed means to separate their components. Nop7p is associated primarily with 66S preribosomes containing either 27SB or 25.5S plus 7S pre-rRNAs. Copurifying proteins identified by mass spectrometry include ribosomal proteins, nonribosomal proteins previously implicated in 60S ribosome biogenesis, and proteins not known to be involved in ribosome production. Analysis of strains mutant for eight of these proteins not previously implicated in ribosome biogenesis showed that they do participate in this pathway. These results demonstrate that proteomic approaches in concert with genetic tools provide powerful means to purify and characterize ribosome assembly intermediates.

Cell Fractionation↗

Overexpression analysis of plant transcription factors.

Transcription factors (TFs) play important roles in plant development and its response to the environment. A variety of reverse genetics tools have been developed to study TF function, the two most commonly used ones being knockout and overexpression. Because of the unique characteristics and modes of action of TFs, the overexpression strategy has been particularly effective in revealing TF function. Thus, a number of overexpression-based methodologies - constitutive expression, tissue-specific expression, chemically inducible expression and overexpression of modified TFs - have been developed and are used in the analysis of TF function.

Gene Expression Regulation, Developmental↗

Gene disruption to evaluate the role of fungal candidate virulence genes.

Gene disruption is a powerful genetic tool that can define pathogenic or virulence factors. In the past two years gene disruption approaches have been used to identify fungal virulence genes. The capsule genes, an alpha subunit of G protein and certain kinases of Cryptococcus neoformans have clearly been demonstrated to be associated with pathogenicity. In Candida albicans at least four genes involved in hyphal formation have been disrupted and tested for virulence. In other fungi, such as Histoplasma capsulatum, however, more efficient gene disruption methods need to be developed before such approaches can be regularly used for identifying virulence genes.

Candida↗

Mouse-based phenogenomics for modelling human disease.

The powerful and wide-ranging genetic tools available in the laboratory mouse make it the major experimental model for studying mammalian gene function in vivo and modelling human disease traits. Large-scale random mutagenesis approaches, either gene-driven or phenotype-driven, promise to identify new clinically relevant phenotypes and their associated genes. Development of appropriate tools for assessing clinical phenotypes in mice is a crucial component of these endeavours, as is the establishment of the infrastructure for archiving and distribution of the growing mutant resource to the community. Integrated, multidisciplinary programs will be needed to fully exploit the power of the mouse in molecular medicine.

Academies and Institutes↗

The role of calcium-activated potassium channels in respiratory control.

The introduction of pharmacological and genetic tools has helped to understand the functional role of specific ion channel subtypes in the neural control of breathing. This review outlines the role one family of potassium channels, those, which are regulated by intracellular calcium. Calcium-activated potassium channels (K(+)ca) play important roles in determining the duration of single action potentials and in the frequency of action potentials in excitable cells. They have been classified based on their unitary conductances combined with their inhibition by a variety of pharmacologic substances. In this review the role of K(+)ca channels in respiratory control is outlined. The predictions of their contributions from modeling the respiratory system and the results of experiments in which they are individually blocked are discussed. Recently mice have been generated in which the expression of a single member of the K(+)ca channel family can be conditionally regulated. The repression of this small-conductance K(+)ca channel (SK3) as well as its overexpression affect baseline respiratory rhythm and the ventilatory response to hypoxia.

Animals↗

The Hermes transposable element from the house fly, Musca domestica, is a short inverted repeat-type element of the hobo, Ac, and Tam3 (hAT) element family.

The genome of the house fly, Musca domestica, contains an active transposable element system, called Hermes. Using PCR and inverse PCR we amplified and sequenced overlapping segments of several Hermes elements and from these data we have constructed a 2749 bp consensus Hermes DNA sequence. Hermes termini are composed of 17 bp imperfect inverted repeats that are almost identical to the inverted terminal repeats of the hobo element of Drosophila melanogaster. Full length Hermes elements contain a single long ORF capable of encoding a protein of 612 amino acids which is 55% identical to the amino acid sequence of the hobo transposase. Comparison of the ends of the Hermes and hobo elements to those of the Ac element of Zea mays, and the Tam3 element of Antirrhinum majus, as well as several other plant and insect elements, revealed a conserved terminal sequence motif. Thus Hermes is clearly a member of the hobo, Ac and Tam3 (hAT) transposable element family, other members of which include the Tag1 element from Arabidopsis thaliana and the Bg element from Zea mays. The evolution of this class of transposable elements and the potential utility of Hermes as a genetic tool in M. domestica and related species are discussed.

Amino Acid Sequence↗