Monitoring microbial predator-prey interactions: an experimental study using fatty acid biomarker and compound-specific stable isotope techniques

Naturally occurring microbial communities are complex, withautotrophs and heterotrophs often similarly sized and impossibleto separate by conventional size fractionation approaches. However,if it was possible to identify specific compounds that are characteristicof particular groups of microbes and determine the stable isotopecomposition of these biomarkers, the requirement for size fractionationcould potentially be negated. This work considered the usefulnessof such an approach by analysis of a simple laboratory predator–preysystem comprising Nanochloropsis oculata, an autotrophic flagellateprey and Oxyrrhis marina, a heterotrophic flagellate predator.In growth-grazing experiments the fatty acids 20:5(n–3)and 22:6(n–3) were used as biomarkers for N. oculata andO. marina respectively. Interpretation of ¹³C values of thesepredator and prey fatty acid biomarkers was not straightforwardsince although isotopic signature of the O. marina biomarkerwas consistently enriched compared to that of its N. oculataprey, the magnitude of enrichment in ¹³C increased with ageof culture (1.0–5.4 %). Given the variability we observedin our experimental cultures, it will be difficult to applythis approach to complex field situations without a comprehensiveunderstanding of the factors determining the ¹³C values of specificbiomarker molecules.     

Methylphenidate exposure induces dopamine neuron loss and activation of microglia in the basal ganglia of mice

Background

Methylphenidate (MPH) is a psychostimulant that exerts its pharmacological effects via preferential blockade of the dopamine transporter (DAT) and the norepinephrine transporter (NET), resulting in increased monoamine levels in the synapse. Clinically, methylphenidate is prescribed for the symptomatic treatment of ADHD and narcolepsy; although lately, there has been an increased incidence of its use in individuals not meeting the criteria for these disorders. MPH has also been misused as a “cognitive enhancer” and as an alternative to other psychostimulants. Here, we investigate whether chronic or acute administration of MPH in mice at either 1 mg/kg or 10 mg/kg, affects cell number and gene expression in the basal ganglia.

Methodology/Principal Findings

Through the use of stereological counting methods, we observed a significant reduction (∼20%) in dopamine neuron numbers in the substantia nigra pars compacta (SNpc) following chronic administration of 10 mg/kg MPH. This dosage of MPH also induced a significant increase in the number of activated microglia in the SNpc. Additionally, exposure to either 1 mg/kg or 10 mg/kg MPH increased the sensitivity of SNpc dopaminergic neurons to the parkinsonian agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Unbiased gene screening employing Affymetrix GeneChip® HT MG-430 PM revealed changes in 115 and 54 genes in the substantia nigra (SN) of mice exposed to 1 mg/kg and 10 mg/kg MPH doses, respectively. Decreases in the mRNA levels of gdnf, dat1, vmat2, and th in the substantia nigra (SN) were observed with both acute and chronic dosing of 10 mg/kg MPH. We also found an increase in mRNA levels of the pro-inflammatory genes il-6 and tnf-α in the striatum, although these were seen only at an acute dose of 10 mg/kg and not following chronic dosing.

Conclusion

Collectively, our results suggest that chronic MPH usage in mice at doses spanning the therapeutic range in humans, especially at prolonged higher doses, has long-term neurodegenerative consequences.     

3-Fluorotyrosine as a Complementary Probe of Hemoglobin Structure and Dynamics: A (19) F-NMR Study of Synechococcus sp. PCC 7002 GlbN

The hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002 (GlbN) contains three tyrosines (Tyr5, Tyr22, and Tyr53), each of which undergoes a structural rearrangement when the protein binds an exogenous ligand such as cyanide. We explored the use of 3-fluorotyrosine and (19) F-NMR spectroscopy for the characterization of GlbN. Assignment of (19) F resonances in fluorinated GlbN (GlbN*) was achieved with individual Tyr5Phe and Tyr53Phe replacements. We observed marked variations in chemical shift and linewidth reflecting the dependence of structural and dynamic properties on oxidation state, ligation state, and covalent attachment of the heme group. The isoelectronic complexes of ferric GlbN* with cyanide and ferrous GlbN* with carbon monoxide gave contrasting spectra, the latter exhibiting heterogeneity and enhanced internal motions on a microsecond-to-millisecond time scale. The strength of the H-bond network involving Tyr22 (B10) and bound cyanide was tested at high pH. 3-Fluorotyrosine at position 22 had a pK(a) value at least 3 units higher than its intrinsic value, 8.5. In addition, evidence was found for long-range communication among the tyrosine sites. These observations demonstrated the utility of the 3-fluorotyrosine approach to gain insight in hemoglobin properties.     

Critical role of acrolein in secondary injury following ex vivo spinal cord trauma

The pathophysiology of spinal cord injury (SCI) is characterized by the initial, primary injury followed by secondary injury processes in which oxidative stress is a critical component. Secondary injury processes not only exacerbate pathology at the site of primary injury, but also result in spreading of injuries to the adjacent, otherwise healthy tissue. The lipid peroxidation byproduct acrolein has been implicated as one potential mediator of secondary injury. To further and rigorously elucidate the role of acrolein in secondary injury, a unique ex vivo model is utilized to isolate the detrimental effects of mechanical injury from toxins such as acrolein that are produced endogenously following SCI. We demonstrate that (i) acrolein-Lys adducts are capable of diffusing from compressed tissue to adjacent, otherwise uninjured tissue; (ii) secondary injury by itself produces significant membrane damage and increased superoxide production; and (iii) these injuries are significantly attenuated by the acrolein scavenger hydralazine. Furthermore, hydralazine treatment results in significantly less membrane damage 2 h following compression injury, but not immediately after. These findings support our hypothesis that, following SCI, acrolein is increased to pathologic concentrations, contributes significantly to secondary injury, and thus represents a novel target for scavenging to promote improved recovery.     

Raccoon ecology database: A resource for population dynamics modelling and meta-analysis

The value of scientific studies increases and is extended when their data are stored in a manageable and accessible format. This is demonstrated through development of a raccoon ecology database (REDB) to store, manage and disseminate available peer-reviewed and unpublished data on raccoon (Procyon lotor) biology, ecology and raccoon rabies, including citations for data sources. Over 800 documents were identified and citations for them entered into the database as literature references. Approximately 1000 trait values were entered from almost 200 of these sources. These data included estimates of population density, survival rates, rabies incubation period, litter size, body weight, dispersal distance and home-range size, often by age or sex class. Each datum is linked to a citation for its source, and to information about location and land use in the study area, time of year the study was undertaken, sample size, and variance. The relational database design enables querying and easy updating and manipulation of data.

The relational data model is presented, as is its application in further developing an individual-based, spatially-explicit population model of raccoon rabies. Using information queried from the REDB benefits model development by: i) assessing the appropriateness of input parameter values, ii) providing sources for citing input values, iii) parameterising the model to different geographic regions, iv) enabling meta-analyses for evaluating model structure, as well as further contributing to parameterisation at specific locations, and v) providing biologically appropriate parameter input values for model sensitivity testing. The REDB is a useful research resource that will increase in value with ongoing inclusion of data from future raccoon and raccoon rabies studies and serves as a model for database design and research applications to other species. The database and an empty database for use with other species are available online (http://redb.nrdpfc.ca).     

Pretreatment with drug-specific antibody reduces desipramine cardiotoxicity in rats

The effect of a drug-specific antibody on desipramine (DMI) cardiotoxicity was studied in rats. Animals were pretreated i.v. with 4.2 g/kg of a monoclonal antibody (anti-TCA) followed by DMI HCl 30 mg/kg i.p. (molar ratio of anti-TCA binding sites to DMI = 0.56). Peak QRS complex prolongation was substantially lower after pretreatment with anti-TCA than after control antibody (70 +/- 14 v. 21 +/- 4%, p less than 0.001). Time to peak toxicity was the same in both groups. Binding of DMI by anti-TCA was demonstrated by a higher serum total DMI concentration and increased DMI binding in serum after anti-TCA compared to controls. The DMI concentration in anti-TCA treated animals was lower in some organs (brain, lung, liver, spleen), but not in others (heart, muscle, kidney, fat). The calculated fraction of the DMI dose bound by anti-TCA was 19.9%. The steepness of the DMI dose-response curve was examined by administering DMI alone (without antibody) at various doses to rats. Compared to 30 mg/kg DMI, a dose reduction of 30-50% was needed to reduce QRS duration to the same extent as anti-TCA pretreatment. We conclude that DMI cardiotoxicity was markedly reduced by the binding of a relative small fraction of the DMI body burden to anti-TCA. This disproportionate effect of DMI binding was not due to the steepness of the DMI dose-response curve, nor to slowing of the rate of DMI distribution to tissues.     

Hydrostatic Pressure and Temperature Effects on the Membranes of a Seasonally Migrating Marine Copepod

Marine planktonic copepods of the order Calanoida are central to the ecology and productivity of high latitude ecosystems, representing the interface between primary producers and fish. These animals typically undertake a seasonal vertical migration into the deep sea, where they remain dormant for periods of between three and nine months. Descending copepods are subject to low temperatures and increased hydrostatic pressures. Nothing is known about how these organisms adapt their membranes to these environmental stressors. We collected copepods (Calanoides acutus) from the Southern Ocean at depth horizons ranging from surface waters down to 1000 m. Temperature and/or pressure both had significant, additive effects on the overall composition of the membrane phospholipid fatty acids (PLFAs) in C. acutus. The most prominent constituent of the PLFAs, the polyunsaturated fatty acid docosahexanoic acid [DHA – 22:6(n-3)], was affected by a significant interaction between temperature and pressure. This moiety increased with pressure, with the rate of increase being greater at colder temperatures. We suggest that DHA is key to the physiological adaptations of vertically migrating zooplankton, most likely because the biophysical properties of this compound are suited to maintaining membrane order in the cold, high pressure conditions that persist in the deep sea. As copepods cannot synthesise DHA and do not feed during dormancy, sufficient DHA must be accumulated through ingestion before migration is initiated. Climate-driven changes in the timing and abundance of the flagellated microplankton that supply DHA to copepods have major implications for the capacity of these animals to undertake their seasonal life cycle successfully.