Interactions of Carbon Dioxide and Food Odours in Drosophila: Olfactory Hedonics and Sensory Neuron Properties

Behavioural responses of animals to volatiles in their environment are generally dependent on context. Most natural odours are mixtures of components that can each induce different behaviours when presented on their own. We have investigated how a complex of two olfactory stimuli is evaluated by Drosophila flies in a free-flying two-trap choice assay and how these stimuli are encoded in olfactory receptor neurons. We first observed that volatiles from apple cider vinegar attracted flies while carbon dioxide (CO₂) was avoided, confirming their inherent positive and negative values. In contradiction with previous results obtained from walking flies in a four-field olfactometer, in the present assay the addition of CO₂ to vinegar increased rather than decreased the attractiveness of vinegar. This effect was female-specific even though males and females responded similarly to CO₂ and vinegar on their own. To test whether the female-specific behavioural response to the mixture correlated with a sexual dimorphism at the peripheral level we recorded from olfactory receptor neurons stimulated with vinegar, CO₂ and their combination. Responses to vinegar were obtained from three neuron classes, two of them housed with the CO₂-responsive neuron in ab1 sensilla. Sensitivity of these neurons to both CO₂ and vinegar per se did not differ between males and females and responses from female neurons did not change when CO₂ and vinegar were presented simultaneously. We also found that CO₂-sensitive neurons are particularly well adapted to respond rapidly to small concentration changes irrespective of background CO₂ levels. The ability to encode temporal properties of stimulations differs considerably between CO₂- and vinegar-sensitive neurons. These properties may have important implications for in-flight navigation when rapid responses to fragmented odour plumes are crucial to locate odour sources. However, the flies’ sex-specific response to the CO₂-vinegar combination and the context-dependent hedonics most likely originate from central rather than peripheral processing.     

Time Course of Adaptations in Dopamine Biosynthesis, Metabolism, and Release Following Nigrostriatal Lesions: Implications for Behavioral Recovery from Brain Injury

Alterations in neostriatal dopamine metabolism, release, and biosynthesis were determined 3, 5, or 18 days following partial, unilateral destruction of the rat nigrostriatal dopamine projection. Concentrations of dopamine and each of its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3-methoxytyramine (3-MT) were markedly decreased in the lesioned striata at 3, 5, or 18 days postoperation. The decline in striatal high-affinity [3H]dopamine uptake closely matched the depletion of dopamine at 3 and 18 days postoperation. However, neither DOPAC, HVA, nor 3-MT concentrations were decreased to as great an extent as dopamine at any time following lesions that depleted the dopamine innervation of the striatum by greater than 80%. In these more severely lesioned animals, dopamine metabolism, estimated from the ratio of DOPAC or HVA to dopamine, was increased two- to four-fold in the injured hemisphere compared with the intact hemisphere. Dopamine release, estimated by the ratio of 3-MT to dopamine, was more increased, by five- to sixfold. Importantly, the HVA/dopamine, DOPAC/dopamine, and 3-MT/dopamine ratios did not differ between 3 and 18 days postlesioning. The rate of in vivo dopamine biosynthesis, as estimated by striatal DOPA accumulation following 3,4-dihydroxyphenylalanine (DOPA) decarboxylase inhibition with NSD 1015, was increased by 2.6- to 2.7-fold in the surviving dopamine terminals but again equally at 3 and 18 days postoperation. Thus, maximal increases in dopamine metabolism, release, and biosynthesis occur rapidly within neostriatal terminals that survive a lesion. This mobilization of dopaminergic function could contribute to the recovery from the behavioral deficits of partial denervation by increasing the availability of dopamine to neostriatal dopamine receptors. However, these presynaptic compensations are not sufficient to account for the protracted (at least 3-week) time course of sensorimotor recovery that has been observed following partial nigrostriatal lesion.     

Detection of ornamental transgenic fish by real-time PCR and fluorescence microscopy

Transgenic Research - Numbers of ornamental transgenic fish are increasing, and some of them are illegally imported into Europe. The fish are modified to display different fluorescent colors under...     

Seeing the Quiet Politics in Unquiet Woods: A Different Vantage Point for a Future Forest Agenda

Human Ecology - We address two aspects of forest lives—violence and care—that are central to forest outcomes but often invisible in mainstream discussions on forests. We argue that...     

Insights in KIR2.1 channel structure and function by an evolutionary approach; cloning and functional characterization of the first reptilian inward rectifier channel KIR2.1, derived from the California kingsnake (Lampropeltis getula californiae)

Potassium inward rectifier K_IR2.1 channels contribute to the stable resting membrane potential in a variety of muscle and neuronal cell-types. Mutations in the K_IR2.1 gene KCNJ2 have been associated with human disease, such as cardiac arrhythmias and periodic paralysis. Crystal structure and homology modelling of K_IR2.1 channels combined with functional current measurements provided valuable insights in mechanisms underlying channel function. K_IR2.1 channels have been cloned and analyzed from all main vertebrate phyla, except reptilians. To address this lacuna, we set out to clone reptilian K_IR2.1 channels. Using a degenerated primer set we cloned the KCNJ2 coding regions from muscle tissue of turtle, snake, bear, quail and bream, and compared their deduced amino acid sequences with those of K_IR2.1 sequences from 26 different animal species obtained from Genbank. Furthermore, expression constructs were prepared for functional electrophysiological studies of ectopically expressed K_IR2.1 ion channels. In general, KCNJ2 gene evolution followed normal phylogenetic patterns, however turtle K_IR2.1 ion channel sequence is more homologues to avians than to snake. Alignment of all 31 K_IR2.1 sequences showed that all disease causing K_IR2.1 mutations, except V93I, V123G and N318S, are fully conserved. Homology models were built to provide structural insights into species specific amino acid substitutions. Snake K_IR2.1 channels became expressed at the plasmamembrane and produced typical barium sensitive (IC₅₀ ∼6 μM) inward rectifier currents.     

Opposing Roles of Dnmt1 in Early- and Late-Stage Murine Prostate Cancer

Previous studies have shown that tumor progression in the transgenic adenocarcinoma of mouse prostate (TRAMP) model is characterized by global DNA hypomethylation initiated during early-stage disease and locus-specific DNA hypermethylation occurring predominantly in late-stage disease. Here, we utilized Dnmt1 hypomorphic alleles to examine the role of Dnmt1 in normal prostate development and in prostate cancer in TRAMP. Prostate tissue morphology and differentiation status was normal in Dnmt1 hypomorphic mice, despite global DNA hypomethylation. TRAMP; Dnmt1 hypomorphic mice also displayed global DNA hypomethylation, but were characterized by altered tumor phenotype. Specifically, TRAMP; Dnmt1 hypomorphic mice exhibited slightly increased tumor incidence and significantly increased pathological progression at early ages and, conversely, displayed slightly decreased tumor incidence and significantly decreased pathological progression at advanced ages. Remarkably, hypomorphic Dnmt1 expression abrogated local and distant site macrometastases. Thus, Dnmt1 has tumor suppressor activity in early-stage prostate cancer, and oncogenic activity in late stage prostate cancer and metastasis. Consistent with the biological phenotype, epigenomic studies revealed that TRAMP; Dnmt1 hypomorphic mice show dramatically reduced CpG island and promoter DNA hypermethylation in late-stage primary tumors compared to control mice. Taken together, the data reveal a crucial role for Dnmt1 in prostate cancer and suggest that Dnmt1-targeted interventions may have utility specifically for advanced and/or metastatic prostate cancer.Changes in DNA methyltransferase (Dnmt) expression and DNA methylation are observed in human prostate cancer (3, 38, 41). Of particular interest, genes with tumor suppressive function become hypermethylated and silenced, which correlates with the development of specific disease phenotypes (2, 3, 38). Although an association between prostate cancer and alterations in DNA methylation has been established, in vivo models are required to determine whether these changes functionally contribute to the disease. In this context, studies in which pharmacological inhibitors of Dnmts were shown to inhibit prostate cancer in murine models have proven informative (34, 56). However, it remains unknown whether genetic disruption of epigenetic components, such as Dnmts, also impacts prostate cancer development. This is a critical question since the pharmacological inhibitors of Dnmts have pleiotropic effects, including those unrelated to activation of methylation-silenced genes (21, 23, 31). Moreover, no studies to date have examined whether Dnmts or DNA methylation play roles in normal prostate development; this information is vital to fully understanding the effects that inhibiting DNA methylation may have on prostate cancer.Dnmt1 is a maintenance DNA methyltransferase that propagates preexisting DNA methylation patterns in genomic DNA (44). Dnmt1 also is involved in de novo DNA methylation in cancer cells and interacts with other key epigenetic control molecules, including histone-modifying enzymes (11, 19). Murine models have been used to investigate the in vivo functions of Dnmt1. Complete genetic knockout of Dnmt1 is embryonic lethal in mice (29). However, hypomorphic expression of Dnmt1 allows murine development to proceed but causes global DNA hypomethylation and impacts cancer development and progression (7, 14, 28). Specifically, hypomorphic expression of Dnmt1 can lead to the development of lymphoma (14). Furthermore, crossing Dnmt1 hypomorphic mice with murine tumor models alters tumor progression, resulting in either increased or decreased tumor development, depending on the disease stage and tissue site (1, 7, 53). For example, reduced expression of Dnmt1 dramatically decreases intestinal polyp formation in Apc^Min/+ mice, either alone or in combination with 5-aza-2′-deoxycytidine treatment (7, 27). However, it was later noted that reduced expression of Dnmt1 has a dual effect on intestinal cancer in Apc^Min/+ mice, in which the development of early stage intestinal microadenomas is accelerated, whereas the formation of adenomatous polyps is significantly reduced (53). In addition, Apc^Min/+ Dnmt1 hypomorphic mice develop liver cancer associated with the loss of heterozygosity of Apc (53). Similarly, in Dnmt1 hypomorphic mice crossed to Mlh1^−/− mice, a dual effect was noted wherein mice developed fewer intestinal cancers but displayed increased T- and B-cell lymphomas (52). In addition, a recent study demonstrated that hypomorphic Dnmt1 expression is associated with reduced squamous cell carcinoma of the tongue and esophagus, resulting in decreased invasive cancer (1). Taken together, the data suggest that Dnmt1 has diverse effects on cancer development, which are dependent on tissue context and tumor stage.TRAMP is a well-established transgenic prostate cancer model driven by prostate-specific expression of the simian virus 40 (SV40) T/t oncogenes (16). TRAMP mice are characterized by Dnmt mRNA and protein overexpression, altered DNA methylation, and altered gene expression during prostate cancer development (2, 33, 35, 37). Of the three enzymatically active Dnmts, Dnmt1 shows the greatest level of overexpression in TRAMP, and this correlates with Rb inactivation, a key genetic event driving prostate cancer in the model (37). Most critically, global DNA hypomethylation occurs during early and late disease stages, while DNA hypermethylation occurs primarily at late disease stages in TRAMP (35).Here, we utilized Dnmt1 hypomorphic mice and the TRAMP model to assess the role of DNA methylation in both normal prostatic development and prostate cancer. The Dnmt1 hypomorphic mouse model used involves two different hypomorphic alleles (N and R), resulting in four genotypes with progressively reduced DNA methylation (Dnmt1^+/+, Dnmt1^R/+, Dnmt1^N/+, and Dnmt1^N/R) (7, 52). The N allele consists of a PGK-Neo insertion that deletes a portion of exon 4 of Dnmt1, resulting in severely reduced Dnmt1 expression, while the R allele involves a lacO insertion into intron 3 of Dnmt1, which partially reduces Dnmt1 expression (7, 52). Based on our previous work establishing the timing of DNA hypomethylation and DNA hypermethylation in TRAMP, we hypothesized that hypomorphic Dnmt1 expression in TRAMP may have tumor-promoting effects at early disease stages and tumor-inhibitory effects at later stages of prostate cancer progression. Our data are consistent with this hypothesis and, more importantly, reveal a critical and unanticipated role for Dnmt1 in prostate cancer metastasis.     

A thioesterase bypasses the requirement for exogenous fatty acids in the plsX deletion of Streptococcus pneumoniae

PlsX is an acyl‐acyl carrier protein (ACP):phosphate transacylase that interconverts the two acyl donors in Gram‐positive bacterial phospholipid synthesis. The deletion of plsX in Staphylococcus aureus results in a requirement for both exogenous fatty acids and de novo type II fatty acid biosynthesis. Deletion of plsX (SP0037) in Streptococcus pneumoniae did not result in an auxotrophic phenotype. The ΔplsX S. pneumoniae strain was refractory to myristic acid‐dependent growth arrest, and unlike the wild‐type strain, was susceptible to fatty acid synthesis inhibitors in the presence of exogenous oleate. The ΔplsX strain contained longer chain saturated fatty acids imparting a distinctly altered phospholipid molecular species profile. An elevated pool of 18‐ and 20‐carbon saturated fatty acids was detected in the ΔplsX strain. A S. pneumoniae thioesterase (TesS, SP1408) hydrolyzed acyl‐ACP in vitro, and the ΔtesS ΔplsX double knockout strain was a fatty acid auxotroph. Thus, the TesS thioesterase hydrolyzed the accumulating acyl‐ACP in the ΔplsX strain to liberate fatty acids that were activated by fatty acid kinase to bypass a requirement for extracellular fatty acid. This work identifies tesS as the gene responsible for the difference in exogenous fatty acid growth requirement of the ΔplsX strains of S. aureus and S. pneumoniae.     

Progressive sensorimotor impairment is not associated with reduced dopamine and high energy phosphate donors in a model of ataxia-telangiectasia

Ataxia-telangiectasia (A-T) is a genetic disease, associated with progressive motor impairment and a lack of functional ATM protein. It has been reported that immunoreactive tyrosine hydroxylase and dopamine transporter are reduced in an Atm-/- mouse model of A-T. These observations led to a hypothesis that A-T is associated with loss of nigrostriatal dopamine and prompted the launch of clinical trials to evaluate a therapeutic utility of the anti-parkinsonian drug, l-DOPA. To test for dopamine depletion more directly, we measured regional levels of monoamines and their metabolites in the Atm-/- mouse brain. We also measured levels of NAD+, a cofactor for dopamine biosynthesis and substrate of the DNA damage surveillance enzyme, poly(ADP-ribose) polymerase (PARP). Constitutive activation of PARP has been posited to cause NAD+ depletion. We observed no reduction in monoamine transmitters and no depletion of NAD+, or other high energy phosphate donors in the adult Atm-/- cerebellum, striatum, or ventral mesencephalon. However, our studies did reveal a progressive sensorimotor impairment in Atm-/- mice that may serve as a relevant proxy for progressive neurological impairment in the human disease. Our results call into question the involvement of dopamine in A-T and the therapeutic strategy of enhancing dopaminergic function with l-DOPA.     

Occurrence of 5-methoxypodophyllotoxin in plants,cell cultures and regenerated plants of Linum flavum

The 5-methoxypodophyllotoxin contents of plants, cell cultures and regenerated plants of Linum flavum are compared. It is demonstrated that cell cultures are able to produce amounts of 5-methoxypodophyllotoxin that are comparable to the concentration in fully differentiated plants. The production of 5-methoxy-podophyllotoxin depends on the hormonal balance of the growth medium. The use of 2,4-dichlorophenoxyacetic acid as the growth regulator is favourable for 5-methoxypodophyllotoxin production when compared to naphthylacetic acid. The 5-methoxypodophyllotoxin accumulation appears to be positively related to the internal cell volume.